CN107805277A - Sepiella maindroni Semen sarameters and preparation method and purposes - Google Patents

Abstract

The invention discloses a surface protein of squid mansoni sperm and its preparation method and application. For the first time, the full-length cDNA sequence of the surface protein Sp17 of squid mansoni is cloned, and the physical and chemical analysis is performed according to the amino acid sequence of the cDNA. properties and construct phylogenetic trees. The expression specificity of different tissues was analyzed for the surface protein Sp17 of squid mansoni sperm by fluorescence quantitative PCR method. The beneficial effects are: the surface protein Sp17 of squid mansoni prepared by the present invention plays an important role in the generation and formation of squid mansoni sperm, and participates in the acrosome reaction to promote the fertilization process, and has a good effect on the spermatozoa mansoni. It has a significant impact on the reproduction of squid mansoni, and it can be used for the regulation of artificial reproduction of squid mansoni. The surface protein Sp17 of manson's needleless cuttlefish sperm can also be used in the detection and treatment of cancer.

Description

Manson's needleless cuttlefish sperm surface protein and its preparation method and use

technical field

The invention relates to the field of genetic engineering, in particular to a sperm surface protein of the squid mansoni, its preparation method and application.

Background technique

During the sexual reproduction of organisms, sperm and egg cells need to fuse together to form a fertilized egg and gradually develop into a new individual. As a highly differentiated haploid cell, sperm cells are different from somatic cells in structure and function. Notably, its function in the process of fertilization is the result of the specific expression of various related genes during sperm development and after maturation, and the development and maturation of sperm cells in terms of structure and function is a highly ordered and quite complex process. A variety of proteins, ions and other biological factors play a role in this process.

Contents of the invention

One of the objects of the present invention is to provide a squid sperm surface protein, and use molecular biology techniques to detect the influence of the squid sperm surface protein on the production and fertilization process of squid sperm.

The second object of the present invention is to provide a method for preparing the sperm surface protein of squid mansoni with good reproducibility and high yield.

The third object of the present invention is to provide a use of the sperm surface protein of squid mansoni, the use of the squid squid sperm surface protein in artificial regulation of squid mansoni reproduction and cancer detection and treatment.

Aiming at the problems mentioned in the background technology, the present invention adopts the following technical scheme: the surface protein of squid mansoni sperm, the full length of cDNA is 1463bp, the 5' non-coding region (UTR) is 92bp, and the 3' non-coding region (UTR) is 222bp , the predicted open reading frame (ORF) is 1149bp in total, encoding 382 amino acids, the relative molecular mass (MW) of the encoded protein is 42.058kDa, and the isoelectric point ( pI ) is 4.66.

The preparation method of the sperm surface protein of the squid mansoni comprises the cloning of the Sp17 gene of the sperm surface protein of the squid mansoni and the specificity analysis of the tissue expression.

The cloning of the Sp17 gene of the sperm surface protein of squid mansoni includes RNA extraction: the testis tissue of sexually mature male squid mansoni is taken, and the total RNA solution is extracted by the Trizol method; RNA quality inspection: the total RNA solution is measured by a nucleic acid protein detector The absorption wavelength at 260nm and 280nm, the value of 260/280 is between 1.8 and 2.0 is qualified; the synthesis of the first strand of cDNA: use the qualified total RNA liquid as a template, and use the reverse transcription kit to carry out the first strand of cDNA Synthesis; specific sample addition system and reaction conditions are as follows:

Add the following reaction system to a 200ml microcentrifuge tube:

Total RNA 2µl

Oligo dt 1µl

DEPC water 3µl

After mixing, centrifuge and place in a PCR instrument, set the temperature at 70°C, take it out immediately after 10 minutes of reaction, and end the ice bath for 2 minutes; continue to add the following reagents to the above reaction system:

RNase Inhibitor (40U/l) 0.25µl

dNTP Mixture (10mM) 0.5µl

M-MLV Rtase (200U/l) 0.25µl

5×M-MLV Buffer 2.0µl

H ₂ O (RNase ^- ) 7.0 µl

Mix well and centrifuge. Set the reaction conditions in the PCR instrument as 42°C for 60 minutes and 70°C for 15 minutes. After the end, take it out quickly, put it in an ice bath for 15 minutes, and store it at -20°C for later use. For long-term storage, place it in a -80°C refrigerator.

The cloning of the Sp17 gene of the sperm surface protein of squid mansoni includes the amplification of the core sequence: using the cDNA of the testis tissue of the mature male squid mansoni as the template, the product fragments of different lengths were amplified by using primers respectively. The reaction system involved is as follows:

2×Es Taq MasterMix (Dye) 12.5µl

Primer F 0.5µl

Primer R 0.5 µl

cDNA 0.5µl

H ₂ O 11 µl

PCR reaction conditions: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycles of reaction; 72°C for 7min; 12°C for 5min; after the reaction, the PCR amplification products were electrophoresed on 1% agarose gel Detect, purify and recover the PCR product with the target band; the primers and their sequences used are: Sp17-1F: AGCAGATGTTAGCCCTTT; Sp17-1R: CTTCAGTATCTTTGGTGCTT; Sp17-2F: TCTTCTAGAGGGTTTCGC; Sp17-2R: TGCAGGAGCTTTTACTTC; Sp17-3F: ACTAAAACTGCTGAAGAG ;Sp17-3R:TGTAGCAGCAGCACTGACCT.

The cloning of the sperm surface protein Sp17 gene of squid squid mansoni includes 5' and 3' RACE amplification of squid squid mansoni Sp17 gene: using 3' RACE and 5' RACE technology to amplify the Sp17 gene of squid squid mansoni; The primers used for the 3'RACE of the Sp17 gene are: 3'-Sp17-outter: AGCAAGATCCAGGCAAGTTTTAGAGGTCA; 3'-Sp17-inner: ATGCTATCGACATTGACCTAACTGACCCA; 3'RACE Adapter: GCGAGCACAGAATTAATATTTTTTTTTTTT; Outter: GCTCCGAACTGAATGA; 5'-Sp17-inner: TTTTGGCTGAGCCCGTAG; 5'RACE Adapter: GGCCAGGCGTCGACTAGTACGGGGGGGGGG.

The cloning of Sp17 gene of needleless cuttlefish sperm surface protein includes full-length cDNA splicing: the verified Sp17 core sequence and 3' and 5' end sequences are spliced to obtain the full-length cDNA sequence of the gene, and the physical alignment is performed according to the amino acid sequence of the cDNA. , chemical properties were predicted and a phylogenetic tree was constructed.

The cDNA of Sp17 gene of sperm surface protein of needleless cuttlefish is:

TGTGACCGGAGAGACTGTCGAGCACGGAATAACACGTGTACCTTGCTATTGAACTAAAGGACAAAGTTAATTCTTCGTTTTGTAAGTTAATCATGTCTGTACCCTTTTCAAACACAAAGTTGAGGGTACCCAGAGGTTTTGAGGCTCTTCTAGAGGGTTTCGCTCGTGAAGTTCTACGGGCTCAGCCAAAATGTATCATTCAGTTCGGAGCCATGCATTTCTCCAATCTTCTCAAGATTCGAACAGAAACAGGCCAAGATCCTGTTGAAGAGTGTGCTGATTTGGAAGATAGGTTCTATAACAATGATTCATTCAAGCATGAAGCTCAAGCAGATGTTAGCCCTTTTGTTGCAGAGACACAGATGGAAGCTCATGCCAGTGAAGAAGTAAAAGCTCCTGCAGAAGAGAAAGTGACTGATAATGCTCAAGAGGAAATTTCACCCAGTGACGTCACAAATGATGAAAATATTTCTGATGCTGCTACAAAGATTCAAGCTGGGTTTAAAGGATACAAGGTTCGAAAAGAAATGAAAGAACGCAAGACTGAACATTCTGAAGAGAAAACGACTGCAGGTACTATAGAAGATGCTGAAGGCACAAAAGATGCTGAAGGCACTAAAACTGCTGAAGAGGCTGCAAATGCTGAAAGCACCAAAGATACTGAAGAGGTTAAAAATGCAGAAGACACTGGAGATGCAGCAGATAATACAAAAGAACAAGAAATACCCAATGAAGAGGTTATAGATATTGACCTCAATGACCCAGAAGTGGCAAACGCTGCTAGCAAGATCCAGGCAAGTTTTAGAGGTCATAAAACCAGGAGAGATCTGCTTAGTAAGCAACAATCAGAACATCTAGAAAATGAAAAAGATGCTAACAACAGTGCAATTACAGAAGACAATGCTATCGACATTGACCTAACTGACCCAGAGGTCAGTGCTGCTGCTACAAAAATTCAAGCAATTTTTCGTGGTCATCAGARCAGGCAAAAACTTAAAGA CACTCCTAAGGATCCAGCAAGTGAGAAAACCATGTCTCAAAAATCTGATAGTGCCACACCTGTTCAAGATGCTACTGGTGACCAGGGACAGGAGGATGACAAGTCTATCAACTATGAGGATCCACAGGTCCAACTGGCTGCCACTAAAATCCAAGCTGGCTTTAAAGGCTACCAAACCCGCAAGAGCCTAAAGAAGCAAGCTGGGAATTCAGAGGATCTTGAAAAGGACAGTGGATCCTAACAAGTTGGTGGATGAAAACAGATGGTGTCTTGATGGAAGAATCCTCTGTTCCTCAGTAAAGCTGATAAAATTTATTGTTCTGATATAATTATTTGTATTGATAAAATAACTCTGGTCCTATTCTTATTGGCTTTCTCCCCCTTCATAGTAAGGGAAATGTTCTGGCAAAGATAAATTTCTTATTTTCTTTAGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA。

The analysis of tissue expression specificity includes extraction of total RNA from each tissue and reverse transcription: extract the brain, optic lobe, muscle, stomach, gills, liver, heart, testis, vas deferens, seminal vesicle, The total RNA of prostate, spermatophore, intestine and pancreas was reverse transcribed to prepare the cDNA template required for real-time PCR experiments; before reverse transcription, the interference of genomic DNA in the extracted total RNA to real-time PCR experiments should be removed, specifically Proceed as follows:

Add the following reaction system to the 200ml centrifuge tube:

Total RNA 1ng

5×gDNA Eraser Buffer 2µl

gDNA Eraser 1µl

Water (Rnase ^- ) 8µl

Mix well and centrifuge for a while, place the centrifuge tube in a PCR instrument, set the reaction conditions as follows: 42°C for 2 minutes, take it out quickly after the reaction and place it in an ice bath for 2 minutes.

Tissue expression specific analysis includes real-time PCR primer design: design primers for real-time PCR based on the obtained Sp17 gene full-length cDNA sequence, specifically: Sp17-YF: TCATTCAGTTCGGAGCCA; Sp17-YR: CCTATCTTCCAAATCAGCACA; Actin-F : TGAGAGGGAGATTGTGCGTG; Actin-R: GAACATAGATTCTGGAGCACGG.

The analysis of tissue expression specificity includes fluorescent quantitative PCR: using the relative fluorescent quantitative method, the expression of Sp17 gene in muscle was selected as a reference, and the squid squid β-actin gene ( S.japonica JN564496.1) was used as an internal reference gene, The expression of Sp17 gene was compared in 14 different tissues of squid squid brain, optic lobe, muscle, stomach, gill, liver, heart, testis, vas deferens, seminal vesicle, prostate, spermatophore, intestine and pancreas, and Perform a significant difference analysis.

Squid mansoni sperm surface protein for use in improving reproductive regulation and cancer detection and treatment of squid mansoni.

Compared with the prior art, the present invention has the advantages of cloning the full-length cDNA sequence of the sperm surface protein Sp17 of the needleless cuttlefish for the first time, predicting its physical and chemical properties and constructing a phylogenetic tree according to the amino acid sequence of the cDNA . The expression specificity of different tissues was analyzed for the surface protein Sp17 of squid mansoni sperm by fluorescence quantitative PCR method. The surface protein Sp17 of squid mansoni prepared by the invention plays an important role in the occurrence and formation of squid mansoni sperm, and participates in the acrosome reaction to promote the fertilization process, and plays a role in the reproduction of squid mansoni Significant impact, it can be used for artificial reproduction regulation of squid mansoni. The surface protein Sp17 of manson's needleless cuttlefish sperm can also be used in the detection and treatment of cancer. The method for preparing the surface protein Sp17 of the squid mansoni sperm of the present invention has good reproducibility and high yield, especially the reverse transcription efficiency is obviously higher than that of the prior art.

Description of drawings

Fig. 1 is the electrophoresis picture extracted from the testis total RNA of Squid Mansoni;

Figure 2 is a graph showing the amplification results of the core fragment of the Sp17 gene;

Fig. 3 is the electrophoresis diagram of the amplification result of the 3' end of the Sp17 gene;

Fig. 4 is the electrophoresis diagram of the amplification result of the 5' end of the Sp17 gene;

Figure 5 is the full-length cDNA of Sp17 gene of squid mansoni;

Figure 6 is a graph of the prediction results of the transmembrane region;

Figure 7 is a graph of the prediction results of the Sp17 signal peptide;

Figure 8 is a comparison diagram of Sp17 amino acid homology;

Figure 9 is an alignment analysis diagram of Sp17 protein domains;

Figure 10 is the secondary structure prediction diagram of Sp17;

Figure 11 is a phylogenetic tree constructed based on the Sp17 homologous amino acid sequence;

Fig. 12 is a graph showing the expression of Sp17 gene in different tissues of squid squid.

Explanation of reference signs: the predicted open reading frame in Figure 5 is marked with a black line frame to mark the initiation codon (ATG) and stop codon (TAA); the same amino acid residues in Figure 8 are marked in black, and the conserved amino acids are marked in gray marked.

Detailed ways

The scheme of the present invention is further described below by accompanying drawing and embodiment:

Example 1:

① Extraction of total RNA from testis tissue

1) Preparations before RNA extraction:

In order to ensure that the RNA extraction process will not be contaminated and cause degradation, before starting to extract RNA, tools such as tweezers and scissors that will directly contact the RNA sample to be extracted should be treated with DEPC, that is, these tools should be soaked overnight in an aqueous solution containing 0.1% DEPC , and sterilized at high temperature for 30 minutes, soaked in absolute ethanol for half an hour before use. The pipette tips and centrifuge tubes used in the experiment must be RNase-free.

2) RNA extraction steps:

a) Add 250ml Trizol reagent to a 1.5ml centrifuge tube, use tweezers and scissors to take about 30 mg of the sample to be extracted stored in the RNA preservation solution and immerse it in Trizol reagent, and quickly use a hand-held electric homogenizer to fully homogenize until the tissue is dispersed. If the liquid is cloudy, add Trizol reagent to a final volume of 1ml;

b) Gently mix the centrifuge tube and let it stand at room temperature for 5 minutes until the tissue is fully lysed;

c) Centrifuge at 12000g for 10min at 4°C;

d) Gently remove the supernatant to avoid agitating the lower layer to precipitate, add it to a new centrifuge tube filled with 200ml pre-cooled chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes;

e) 4°C, 12000g, centrifuge for 15 minutes;

f) After centrifugation, it can be seen that the centrifuge tube is clearly divided into upper, middle and lower layers. Gently take the supernatant and add it to a new pre-cooled centrifuge tube containing 500ml of isopropanol. Mix gently and store at -20°C. Stand for about 1 hour under the condition;

g) 4°C, 12000g, centrifuge for 10min;

h) After centrifugation, a small amount of white precipitate attached to the bottom of the tube can be seen, remove the supernatant, add 1ml of pre-cooled 75% ethanol solution, and blow evenly;

i) 4°C, 7500g, centrifuge for 5min;

j) Remove the supernatant, keep the precipitate, and dry it in the ultra-clean workbench for about 15-20 minutes until the ethanol evaporates to dryness. When the precipitate on the tube wall becomes transparent, add 20ml of DEPC-treated water to dissolve the RNA.

3) Inspection of RNA quality:

The RNA dissolved in DEPC treated water was measured with a nucleic acid protein detector at the absorption wavelengths at 260nm and 280nm. According to relevant data, the 260/280 value between 1.8 and 2.0 indicates that the quality of the extracted RNA is relatively pure, and the impurities contained in it are relatively pure. And the content of polysaccharides is low. RNA below this range is not recommended to be used. If it is higher than 2.0, it means that the extracted RNA is more degraded, and it is not suitable for the next step to amplify the full-length cDNA of the gene. After the measurement is completed, take 2~3ml of the sample and load it on 1% agarose gel electrophoresis. The electrophoresis conditions are set at 135V and 150mA, and the electrophoresis is performed for 15 minutes. The electrophoresis results are obtained by scanning under the gel imaging analysis system, as shown in Figure 1. Take a small amount of dissolved RNA and measure the absorbance at 260nm and 280nm in a nucleic acid and protein detector. The measurement results show that the ratio of A260/A280 is between 1.80 and 2.00, and the electropherogram shows that the 28S band is obvious. The above results indicate that the total RNA extracted from the testis is relatively high in purity, and although there is a small amount of degradation, it is still suitable for the next step of cDNA first-strand synthesis reaction.

② Synthesis of the first strand of cDNA:

In this step, the M-MLV Reverse Transcriptase kit produced by Takara Company was used to synthesize the first strand of cDNA using the total RNA extracted from the testicular tissue of Squid mansoni as a template. The specific loading system and reaction conditions were as follows:

Add the following reaction system to a 200ml microcentrifuge tube:

Total RNA 2µl

Oligo dt 1µl

DEPC water 3µl

Mix well, centrifuge, and place in a PCR instrument, set the temperature at 70°C, take it out immediately after 10 minutes of reaction, and end the ice bath for 2 minutes. Continue to add the following reagents in the above reaction system:

RNase Inhibitor (40U/l) 0.25µl

dNTP Mixture (10mM) 0.5µl

M-MLV Rtase (200U/l) 0.25µl

5×M-MLV Buffer 2.0µl

H ₂ O (RNase ^- ) 7.0 µl

Methyl thiofuroate 0.3µl

Methylcyclopentadiene 0.1µl

Mix well and centrifuge. Set the reaction conditions in the PCR instrument as 42°C for 60 minutes and 70°C for 15 minutes. After the end, take it out quickly, put it in an ice bath for 15 minutes, and store it at -20°C for later use. For long-term storage, place it in a -80°C refrigerator. The methyl thiofuroate and methylcyclopentadiene used in the present invention interact with other components in the reaction system, which can avoid the production of additional reverse transcription products due to the difference in the secondary structure of RNA, and can significantly improve the efficiency of reverse transcription. Increase the activity of transcriptase, improve the efficiency of reverse transcription, and increase the initial concentration of cDNA template.

③ Amplification of the core sequence of the Sp17 gene of squid mansoni:

Using the cDNA of the testis tissue of a mature male squid mansoni as a template, the primers in Table 1 were used to amplify the product fragments of different lengths respectively. The reaction system involved in this process is as follows:

2×Es Taq MasterMix (Dye) 12.5µl

Primer F 0.5µl

Primer R 0.5 µl

cDNA 0.5µl

H ₂ O 11 µl

PCR reaction conditions: 95°C for 5min; 30 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 30s; 72°C for 7min; 12°C for 5min to end;

Table 1 The primers used for cloning the core fragment of Sp17 gene

Tab 1 Primers used for Sp17 gene clone Primer name Sequence (5'to 3') Sp17-1F AGCAGATGTTAGCCCTTT Sp17-1R CTTCAGTATCTTTGGTGCTT Sp17-2F TCTTCTAGAGGGTTTCGC Sp17-2R TGCAGGAGCTTTTACTTC Sp17-3F ACTAAAACTGCTGAAGAG Sp17-3R TGTAGCAGCAGCACTGACCT

After the reaction is completed, the PCR amplification products are detected by 1% agarose gel electrophoresis, and the PCR products with the target bands are purified and recovered. The specific operations are as follows:

a) Prepare 1% agarose gel, electrophoresis after loading the sample, the electrophoresis condition is 100V, 150mA, 20min, after the electrophoresis, the gel is placed in the gel imaging system to scan the electrophoresis band and save it, and cut the target with a clean blade Put the strip in a 1.5ml centrifuge tube (Rnase-), weigh the mass of the strip on an electronic balance, add reagent 1 according to the ratio given in the E.Z.N.A TM Gel Extraction Kit manual, and heat it in a constant temperature water bath at 56°C for 10 minutes. Take it out every 5 minutes and shake it to mix well to speed up the dissolution;

b) Add all the above dissolved mixture into the spin column equipped with the kit, and centrifuge at 10000rpm for 2min;

c) Add all the liquid in the collection tube to the spin column again, and repeat the above operation once;

d) Empty the residual liquid in the collection tube, add 700ml of rinse solution to the spin column, centrifuge at 10000rpm for 1min, and discard the waste liquid;

e) Add 700ml rinse solution to the spin column again and centrifuge;

f) Change to a clean collection tube, evenly add 20ml of EB eluent dropwise on the adsorption membrane, let it stand at room temperature for 2 minutes, centrifuge at 12000rpm for 5 minutes, and collect the recovered target PCR product fragment;

g) Take 1ml of the recovered product to measure its concentration with a nucleic acid and protein detector, and send it to Shanghai Sangon Bioengineering Co., Ltd. for sequencing. After three times of PCR amplification, the target fragments with lengths of 338bp, 256bp and 336bp were respectively obtained, as shown in Figure 2, the samples were sent for sequencing and finally spliced to obtain the core fragment of the squid squid Sp17 gene.

④ RACE amplification of Sp17 gene 3 ^of squid mansoni:

a) Preparation of 3'RACE cDNA template:

Extract the total RNA from the testis tissue of Squid mansoni, and prepare the 3'RACE template cDNA according to the method suggested by the SMART ^™ RACE cDNA Amplification Kit kit. The specific operations are as follows:

Add the following reagents to a 200ml microcentrifuge tube:

Testis total RNA 2µl

3’RACE Adapter 2µl

H ₂ O (Rnase-) 6 µl

Shake and mix the reaction system, centrifuge and place it in a PCR instrument, set the condition at 70°C for 2 minutes, take it out immediately after the reaction, and place it in an ice bath for 2 minutes;

Add the following reagents to the microcentrifuge tube from the previous step:

DTT 2 µl

5×Buffer 4µl

dNTP Mixture (10mM) 2µl

PowerScript Reverse Transcriptase 2µl

Shake and mix the reaction system, centrifuge and place it in a PCR instrument. Set the conditions: 42°C for 1.5 hours, 70°C for 7 minutes. Immediately after the reaction, take out the ice bath for 2 minutes. After the concentration is measured by a nucleic acid and protein detector, dilute to an appropriate concentration. Aliquot and store at -20°C for later use.

b) Amplification of the 3' end of the Sp17 gene:

This process mainly uses nested PCR technology to amplify the 3' end sequence of the gene. The primer sequences involved in this step reaction are shown in the table below. The specific operation is as follows:

Table 2 Primers used in 3'RACE of Sp17 gene

Tab 2 Primers used for Sp17 gene 3'RACE Primer name Sequence (5'to 3') 3'-Sp17-outter AGCAAGATCCAGGCAAGTTTTTAGAGGTCA 3'-Sp17-inner ATGCTATCGACATTGACCTAACTGACCCA 3' RACE Adapter GCGAGCACAGAATTAATATTTTTTTTTTTT

i) Nested round PCR:

Add the following reaction system to the 200ml centrifuge tube:

2×Es Taq MasterMix (Dye) 12.5µl

3'-Sp17-outter 0.5 µl

3’RACE Adapter 0.5µl

cDNA 0.5µl

H ₂ O 11 µl

Mix the system and centrifuge it and place it in a PCR instrument. Set the reaction conditions as follows: 94°C for 5 minutes; 30 cycles at 94°C for 30s, 60°C for 30s, and 72°C for 1 minute; 72°C for 5 minutes; 12°C for 5 minutes.

ii) Nested second-round PCR:

Using a round of PCR product as a template, add the following reaction system to a 200ml centrifuge tube:

2×Es Taq MasterMix (Dye) 12.5µl

3'-Sp17-outter 0.5µl

3’RACE Adapter 0.5µl

One-round PCR product 1.0µl

H ₂ O 10 µl

Mix the reaction system, centrifuge it and place it in a PCR instrument, and set the reaction conditions as follows: 94°C for 5 minutes; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, and react for 35 cycles; 72°C for 5 minutes; 12°C for 5 minutes;

After the reaction, the product was analyzed by 1% agarose gel electrophoresis, the bands are shown in Figure 3, and the target fragment length is 563bp. Cut out the target band for purification and recovery. In order to increase the recovery concentration of PCR products, multiple tubes of PCR products can be mixed together and recovered with ethanol. The specific method: mix according to the volume ratio of PCR products and absolute ethanol at 1:9, and let stand for 5 minutes Then centrifuge at 12000r/min for 5min, discard the supernatant, then wash the precipitate once with 75% ethanol, keep the precipitate at the bottom of the tube after centrifugation, evaporate the ethanol to dryness, and add an appropriate amount of water to dissolve it. The recovered PCR products were connected to the PMD18-T carrier, single bacteria were picked and cultured, and the positive clones verified by bacterial liquid PCR were sent to Shanghai Sangon for sequencing.

⑤ RACE amplification of Sp17 gene 5 ^of squid mansoni:

a) Preparation of 5'RACE template:

Using the total RNA of the testis of squid mansoni as the template, the reverse transcription method is also applicable to the template cDNA preparation of 5'RACE, and the operation steps are as follows:

Add the following reagents to a 200ml centrifuge tube:

Testis total RNA 1.0µl

5'RACE Adapter 1.0µl

Water (Rnase ^- ) 13.5µl

After mixing the reaction system, centrifuge it, place it in a PCR instrument, set the reaction condition to 70°C for 10 minutes, and quickly take it out of the ice bath for 2 minutes after the end.

Add the following reagents to the system where the reaction in the previous step is completed:

dNTP Mixture (10mM) 1.0µl

10×PCR Buffer 2.5µl

0.1M DTT 2.5µl

MgCl ₂ (25mM) 2.5µl

Mix the reaction system and centrifuge, put the centrifuge tube in the PCR instrument, set the reaction conditions as follows: 42°C for 1min; take out and add SuperScript™ II RT 1ml to mix and centrifuge, put it back into the PCR instrument, react at 42°C for 50min, and 70°C for 15min After the end, take it out and add 1ml of RNase mix, put it back, and react at 37°C for 30min.

b) Purification of RACE template cDNA

According to the operation method of the PCR product purification kit, add 5 times the volume of Solution I to the product of the previous reaction, mix well and transfer to the spin column, centrifuge at 10000rpm for 1min, remove the waste liquid in the collection tube, and add 350ml to the spin column For Solution II, centrifuge at 12000rpm for 1min, repeat once; replace with a new collection tube, add 25ml of DEPC-treated water to the spin column, incubate at room temperature for 1min, and finally centrifuge at 12000rpm for 5min to obtain purified cDNA.

c) cDNA tailing, the reaction system is as follows:

Purified cDNA 20µl

2mM dCTP 5µl

5×Tailing Buffer 10µl

Water (RNase-) 13µl

After mixing and centrifuging, the PCR reaction conditions are set as follows: 94°C for 3 minutes, quickly take it out and place it on ice for 2 minutes, add 1ml TdT; 37°C for 10 minutes, and 65°C for 10 minutes to end the reaction, take out the product using a nucleic acid and protein detector Measure the concentration, dilute it with H ₂ O (RNase-) to an appropriate concentration, and store it at -20°C for later use;

d) Amplification of the 5' end of the Sp17 gene:

The 5’RACE method is still carried out using the nested PCR method recommended by the kit. The sequences of the primers involved are shown in the table below. The specific steps are as follows:

Table 3 Primers used in 5'RACE of Sp17 gene

Tab 3 Primers used for sp17 5'RACE Primer name Sequence (5'to 3') 5'-Sp17-outter GCTCCGAACTGAATGA 5'-Sp17-inner TTTTGGCTGAGCCCGTAG 5'RACE Adapter GGCCAGGCGTCGACTAGTACGGGGGGGGGG

i) One round of PCR:

Add the following reagents to a 200ml centrifuge tube:

2×Es Taq MasterMix (Dye) 12.5µl

5'-Sp17-outter 0.5µl

5'RACE Adapter 0.5µl

cDNA 0.5µl

H ₂ O 11 µl

After mixing the reaction system and centrifuging, the centrifuge tube was placed in a PCR instrument, and the reaction conditions were set as follows: 94°C for 5 minutes; 94°C for 30s, 60°C for 30s, 72°C for 30s, 30 cycles of reaction; 72°C for 5min, 12°C for 5min, Take out after the reaction is over;

ii) Second round of PCR:

Add the following reagents to the reaction system:

2×Es Taq MasterMix (Dye) 12.5µl

5'-Sp17-inner 0.5µl

5'RACE Adapter 0.5µl

One-round PCR product 1µl

H ₂ O 11 µl

Mix the reaction system evenly and centrifuge, put the centrifuge tube in the PCR instrument, set the reaction conditions as follows: 94°C for 5 minutes; 94°C for 30s, 60°C for 30s, 72°C for 30s, 35 cycles of reaction; 72°C for 5min, 12°C for 5min , take out after the reaction;

The target band was recovered by electrophoresis of the PCR product after the above reaction, and the product was analyzed by 1% agarose gel electrophoresis. The band was shown in Figure 4, and the target fragment was 210 bp in length. The purified PCR product was connected to the PMD18-T vector and transferred to DH5α competent cells. After plated, a single clone was picked and cultured. The bacterial solution was verified by PCR and sent to Shanghai Sangon for sequencing.

⑥Sp177 gene cDNA full-length and sequence analysis:

The DNAMAN software was used to compare and full-length splicing of the nucleotide sequences obtained by sequencing to obtain the verified Sp17 core sequence and 3' and 5' end sequences, and splicing to obtain the full-length cDNA sequence of the gene. Use the ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) online tool to predict the open reading frame (ORF) position of the gene, and then use DNAMAN software to translate the nucleotide sequence of the ORF region into the corresponding amino acid sequence . The relative molecular mass and isoelectric point of Sp17 were predicted using the online tool Expasy-ProtParam (http://web.expasy.org/protparam/). Amino acid affinity/hydrophobicity analysis was performed using the online tool Expasy-ProtScale (http://web.expasy.org/cgi-bin/protscale/protscale.pl), NetPhos 3.1 Server (http://www.cbs.dtu.dk/ services/NetPhos/) for analysis of amino acid phosphorylation sites, scratch protein predictor (http://scratch.proteomics.ics.uci.edu/) for analysis of disulfide bond positions in amino acid sequences, and protein secondary structure prediction using Antheprot5.0 Software analysis, using the online tool SignaIP (http://www.cbs.dtu.dk/services/SignalP/) to predict the signal peptide of the encoded amino acid sequence, and using TMHMM Server v .2.0 (http://www.cbs. dtu.dk/servi-ces/TMHMM/) for structure prediction of the transmembrane region. Blastx and Blastn analysis of the amino acid sequence of Sp17 were performed using NCBI online tools, and Sp17 and its homologous amino acid sequences were compared with ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalo/), the conserved structure Domain analysis uses NCBI's online search tool Conserved domain search (https://www.ncbi.nlm.nih.gov/Structure/cdd/-wrpsb.cgi). The other 65 Sp17 homologous amino acid sequences selected for phylogenetic analysis were all from the NCBI database, and the MEGA5.0 software was used for alignment and the ClustalW function was used for preliminary processing of these amino acid sequences, and then Phylogeny.fr (http://www.phylogeny.fr. .phylogeny.fr/) The Gblooks function of the online website analyzes the conservative amino acids of the amino acid sequence. After exporting the corresponding results, the Modelgenerator software is used to predict the amino acid substitution model, and the parameters of the evolutionary tree model are determined. Finally, the mrBayes software is used based on Bayesian theory Build a phylogenetic tree. PEST sequence analysis was performed using the online website PEST FIND (http://emboss.bioinformatics.nl/cgibin/emboss/epestfind) tool.

The final spliced squid squid Sp17 gene cDNA sequence has a total length of 1463bp, 92bp in the 5'UTR region, 222bp in the 3'UTR region, and a predicted open reading frame of 1149bp, encoding a total of 382 amino acids (Figure 5). The analysis of the transmembrane region showed that the protein did not contain a transmembrane region structure (Figure 6), and the online signal peptide prediction results showed that Sp17 did not contain a signal peptide (Figure 7), so the protein may be positioned to function outside the cell. The encoded protein has a relative molecular mass of 42.058kDa and an isoelectric point of 4.66, which is a hydrophilic protein. Further analysis found that there were 45 sites in the amino acid sequence of the protein that might undergo phosphorylation modification. Although there were no obvious disulfide bonds, there was a possibility of potential disulfide bond formation at amino acids 34 and 61.

Using ClustalW online analysis (http://www.ebi.ac.uk/Tools/msa/clustalw2/) Sp17 amino acid homology, the comparison results showed that the Sp17 amino acid sequence of squid mansoni and Pacific oyster ( Crassostrea gigas XP_011412976. 1), sea snail ( Aplysia californica XP_012936202.1), California double-spotted octopus ( Octopus bimaculoides XP_014778086.1), smooth double navel snail ( Biomphalaria glabrata XP_013090015.1), purple sea urchin ( Strongylocentrotus purpuratus XP_018.18) and 5 ( Saccoglossus kowalevskii XP_006818979.1) similarities were 36%, 36%, 36%, 31%, 44% and 38%, respectively, and the conservation of some amino acids in evolution is shown in Figure 8. The corresponding amino acid conserved domains are shown in Figure 9. The DD CABYR SP17 domain and at least one IQ domain are contained in the squid squid Sp17 and its homologous proteins, and the DD CABYR SP17 all appear at the same position (12 -50aa).

Through the analysis of the secondary structure of Sp17 protein, it is found that the protein contains 54% helix (Helix), 3% fold (Sheet), 6% turn (Turn) and 38% random coil (Coil) structure, The relative position of each structure on the peptide chain is shown in FIG. 10 .

Selecting 65 amino acid homologous sequences of Sp17 in squid squid and constructing a phylogenetic tree based on the Bayesian method, the results showed that Sp17 in squid squid was closest to the octopus in evolution, followed by Pacific oyster and sea snail Mollusks and echinoderms such as , purple sea urchin, smooth double navel, and purple sea urchin are most distantly related to humans and East African baboons (Figure 11).

⑦ Extraction of total RNA from different tissues of squid mansoni:

According to the above-mentioned extraction method for extracting total RNA from the testis of Squid mansoni, extract samples from 14 tissues including brain, optic lobe, muscle, stomach, gills, liver, heart, testis, vas deferens, seminal vesicle, prostate, seminal pod, intestine, and pancreas. Total RNA and extracted RNA were stored at -20°C for later use after measuring the absorbance at 260/280nm and electrophoresis verification.

⑧Synthesis of the first strand of cDNA and template dilution:

The total RNA extracted in the previous step from different tissues was reverse transcribed to prepare the cDNA template required for the real-time PCR experiment. For the specific method, refer to the instruction manual of the M-MLV Reverse Transcriptase reagent. Before reverse transcription, it is necessary to remove the interference of genomic DNA in the extracted total RNA on the real-time PCR experiment. The specific steps are as follows:

Add the following reaction system to the 200ml centrifuge tube:

Total RNA 1ng

5×gDNA Eraser Buffer 2µl

gDNA Eraser 1µl

Water (Rnase ^- ) 8µl

Mix well and centrifuge for a while, place the centrifuge tube in a PCR instrument, set the reaction conditions as follows: 42°C for 2 minutes, take it out quickly after the reaction and place it in an ice bath for 2 minutes. Next, use the RNA that has been decontaminated by genomic DNA to synthesize the first strand of cDNA. This step requires adding the following reaction system to the centrifuge tube:

Primer script buffer 24µl

Primerscript RT enzyme mix I1µl

Rt primer mix 1µl

Water (Rnase ^- ) 14µl

The reaction system was mixed and centrifuged, and placed in a PCR instrument to set the reaction conditions: 37°C for 15 minutes, 85°C for 5s, after the end, take out the reverse transcription product and quickly place it in an ice bath for 15 minutes. The product was stored in a -20°C refrigerator for later use. Before the computer experiment, the prepared cDNA of different tissues of Squid mansoni needs to be diluted to a concentration of 100ng/ml. This process requires the use of a nucleic acid and protein detector to measure the cDNA concentration and calculate the dilution factor.

⑨real-time PCR primer design:

Primers for real-time PCR were designed, and the β-actin gene of squid mansoni ( S.japonica JN564496.1) was selected as an internal reference gene. There is no complementarity between the designed primers, and no dimer or hairpin structure will be formed. The specific information is:

Table 4 Primers used in real-time quantitative PCR of Squid squid Sp17 gene

Table 4 Primers of Sp17 in S.japonicaused in quantitative real-time PCR Primer name Sequence (5'to 3') Sp17-YF TCATTCAGTTCGGAGCCA Sp17-YR CCTATCTTTCCAAATCAGCACA Actin-F TGAGAGGGAGATTGTGCGTG Actin-R GAACATAGATTCTGGAGCACGG

⑩Fluorescence quantitative PCR:

The method of relative fluorescence quantification was adopted, the expression of Sp17 gene in muscle was selected as a reference, and the β-actin gene ( S.japonica JN564496.1) was used as an internal reference gene to compare the Sp17 gene Differences in expression levels in 14 different tissues including squid brain, optic lobe, testis, and vas deferens. According to the instruction manual of SYBR Permix Ex TaqTM IIKit reagent, the reaction system is as follows:

2×SYBR 10.0µl

Sp17-YF 0.8 µl

Sp17-YR 0.8 µl

cDNA 0.8µl

ROX Reference Dye II 0.4µl

H ₂ O 7.2 µl

The mixed reaction system was centrifuged and then placed in a fluorescent quantitative PCR instrument, with three auxiliary holes for each sample. Set the reaction conditions as follows: 94°C for 30s; 94°C for 5s; 59°C for 30s, 40 cycles of reaction; 55°C~95°C, 2min to end.

The Sp17 gene was detected in 14 tissues including brain, optic lobe, muscle, stomach, intestine, pancreas, heart, liver, gill, testis, vas deferens, prostate, seminal vesicle, and spermatophore by real-time PCR. For tissue expression specific analysis, the expression level of Sp17 gene in muscle was selected as a reference in the experiment. As shown in Figure 12, the Sp17 gene was significantly expressed in the testes and seminal vesicles of Squid mansoni during sexual maturity ( p <0.05), and it also had higher expression levels in the brain, spermatophores, and vas deferens. However, the expression levels in other tissues were extremely low.

The full-length cDNA of the sperm surface protein 17 (Sp17) gene of the needleless cuttlefish is 1463bp, the predicted open reading frame (ORF) is 1149bp, encoding 382 amino acids, 222bp in the 3'UTR region, and 92bp in the 5'UTR region. The predicted relative molecular mass of the encoded protein is 42.058kDa, pI 4.66. Analysis of the signal peptide and transmembrane region of the protein found that it does not have a signal peptide and transmembrane region structure, and is not a protein secreted in cells. The amino acid sequence of the protein contains 45 phosphorylation sites, which are in the cell signal function in the transduction process. The N-terminal DD CABYR SP17 domain and the potential disulfide bond formation sites at the 34th and 61st amino acids are related to the trimer structure formed by the Sp17 protein during the acrosome reaction. Further analysis found that there is a potential PEST site at the C-terminus of its amino acid sequence, which is related to the process that Sp17 protein can be cleaved into smaller molecular weight proteins. The IQ motif on the peptide chain is related to its ability to bind to the glycosyl molecules on the zona pellucida of the egg membrane. The secondary structure analysis found that the helical structure accounted for a large proportion of the entire peptide chain, especially in the IQ domain. Through amino acid homology comparison, it was found that the protein is not conserved in evolution, and has the closest evolutionary relationship with California double-spot octopus, while the highest similarity is only 44%. Finally, through the tissue difference analysis of Sp17 gene expression, we found that Sp17 gene was mainly expressed significantly in the testis and seminal vesicles, and the expression level in the whole gonad was higher than that in other tissues. Therefore, combined with the previous research results, we Speculated that Sp17 plays a role in the formation and maturation of spermatozoa.

The prepared squid squid sperm surface protein is used to improve the reproductive regulation of squid mansoni, and to detect and treat cancer.

Conventional operations in the operation steps of the present invention are well known to those skilled in the art and will not be repeated here.

The embodiments described above have described the technical solutions of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention, and are not intended to limit the present invention. All done within the principle scope of the present invention Any modification, supplement or substitution in a similar manner shall be included within the protection scope of the present invention.

sequence listing

<110> Zhejiang Ocean University

<120> Sperm surface protein of Manson's needleless cuttlefish and its preparation method and use

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 1463

<212>DNA

<213> Sepiella japonica

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tgtgaccgga gagactgtcg agcacggaat aacacgtgta ccttgctatt gaactaaagg 60

acaaagttaa ttcttcgttt tgtaagttaa tcatgtctgt acccttttca aacacaaagt 120

tgagggtacc cagaggtttt gaggctcttc tagagggttt cgctcgtgaa gttctacggg 180

ctcagccaaa atgtatcatt cagttcggag ccatgcattt ctccaatctt ctcaagattc 240

gaacagaaac aggccaagat cctgttgaag agtgtgctga tttggaagat aggttctata 300

acaatgattc attcaagcat gaagctcaag cagatgttag cccttttgtt gcagagacac 360

agatggaagc tcatgccagt gaagaagtaa aagctcctgc agaagagaaa gtgactgata 420

atgctcaaga ggaaatttca cccagtgacg tcacaaatga tgaaaatatt tctgatgctg 480

ctacaaagat tcaagctggg tttaaaggat acaaggttcg aaaagaaatg aaagaacgca 540

agactgaaca ttctgaagag aaaacgactg caggtactat agaagatgct gaaggcacaa 600

aagatgctga aggcactaaa actgctgaag aggctgcaaa tgctgaaagc accaaagata 660

ctgaagaggt taaaaatgca gaagacactg gagatgcagc agataataca aaagaacaag 720

aaatacccaa tgaagaggtt atagatattg acctcaatga cccagaagtg gcaaacgctg 780

ctagcaagat ccaggcaagt tttagaggtc ataaaaccag gagagatctg cttagtaagc 840

aacaatcaga acatctagaa aatgaaaaag atgctaacaa cagtgcaatt acagaagaca 900

atgctatcga cattgaccta actgacccag aggtcagtgc tgctgctaca aaaattcaag 960

caatttttcg tggtcatcag arcaggcaaa aacttaaaga cactcctaag gatccagcaa 1020

gtgagaaaac catgtctcaa aaatctgata gtgccacacc tgttcaagat gctactggtg 1080

accagggaca ggaggatgac aagtctatca actatgagga tccacaggtc caactggctg 1140

ccactaaaat ccaagctggc tttaaaggct accaaacccg caagagccta aagaagcaag 1200

ctgggaattc agaggatctt gaaaaggaca gtggatccta acaagttggt ggatgaaaac 1260

agatggtgtc ttgatggaag aatcctctgt tcctcagtaa agctgataaa atttattgtt 1320

ctgatataat tatttgtatt gataaaataa ctctggtcct attcttattg gctttctccc 1380

ccttcatagt aagggaaatg ttctggcaaa gataaatttc ttaattttctt taggaaaaaa 1440

aaaaaaaaaaaaaaaaaaaaaaa 1463

Claims (10)

1. The surface protein of Squid Sperm, characterized in that its full-length cDNA is 1463bp, the 5' non-coding region (UTR) is 92bp, the 3' non-coding region (UTR) is 222bp, and the predicted open reading frame (ORF) is 1149bp in total , encoding 382 amino acids, the relative molecular mass (MW) of the encoded protein is 42.058kDa, and the isoelectric point ( pI ) is 4.66. 2. The preparation method of the sperm surface protein of the squid mansoni, which is characterized in that it comprises gene cloning of the sperm surface protein Sp17 of the squid mansoni and analysis of tissue expression specificity. 3. the method for preparing the sperm surface protein of squid mansoni according to claim 2, characterized in that: the gene cloning of the sperm surface protein Sp17 of squid mansoni comprises RNA extraction: taking sexually mature male squid mansoni Trizol method was used to extract the total RNA liquid from the testis tissue of the squid; the inspection of RNA quality: the absorption wavelength of the total RNA liquid at 260nm and 280nm was measured by a nucleic acid protein detector, and the value of 260/280 was between 1.8 and 2.0 as qualified; Synthesis of the first strand of cDNA: Use the qualified total RNA solution as a template, and use a reverse transcription kit to synthesize the first strand of cDNA; the specific loading system and reaction conditions are as follows: Add the following reaction system to a 200ml microcentrifuge tube: Total RNA 2µl Oligo dt 1µl DEPC water 3µl After mixing, centrifuge and place in a PCR instrument, set the temperature at 70°C, take it out immediately after 10 minutes of reaction, and end the ice bath for 2 minutes; continue to add the following reagents to the above reaction system: RNase Inhibitor (40U/l) 0.25µl dNTP Mixture (10mM) 0.5µl M-MLV Rtase (200U/l) 0.25µl 5×M-MLV Buffer 2.0µl H ₂ O (RNase ^- ) 7.0 µl Mix well and centrifuge. Set the reaction conditions in the PCR instrument as 42°C for 60 minutes and 70°C for 15 minutes. After the end, take it out quickly, put it in an ice bath for 15 minutes, and store it at -20°C for later use. For long-term storage, place it in a -80°C refrigerator. 4. the preparation method of Sepia mansoni sperm surface protein according to claim 2, is characterized in that: described Squid mansoni sperm surface protein Sp17 gene clone comprises the amplification of core sequence: with mature Mansoni The cDNA of the testis tissue of male individual squid was used as a template, and the product fragments of different lengths were respectively amplified by using primers. The reaction system involved in this process was as follows: 2×Es Taq MasterMix (Dye) 12.5µl Primer F 0.5µl Primer R 0.5 µl cDNA 0.5µl H ₂ O 11 µl PCR reaction conditions: 95°C for 5min; 94°C for 30s, 55°C for 30s, 72°C for 30s, 30 cycles of reaction; 72°C for 7min; 12°C for 5min; after the reaction, the PCR amplification products were electrophoresed on 1% agarose gel Detect, purify and recover the PCR product with the target band; the primers and their sequences used are: Sp17-1F: AGCAGATGTTAGCCCTTT; Sp17-1R: CTTCAGTATCTTTGGTGCTT; Sp17-2F: TCTTCTAGAGGGTTTCGC; Sp17-2R: TGCAGGAGCTTTTACTTC; Sp17-3F: ACTAAAACTGCTGAAGAG ;Sp17-3R:TGTAGCAGCAGCACTGACCT. 5. the method for preparing the sperm surface protein of squid mansoni according to claim 2, characterized in that: said squid mansoni sperm surface protein Sp17 gene clone comprises squid mansoni Sp17 gene 5' and 3'RACE amplification: using 3'RACE and 5'RACE technology to amplify the Sp17 gene of squid mansoni; wherein, the primers used for the 3'RACE of the Sp17 gene are: 3'-Sp17-outter: AGCAAGATCCAGGCAAGTTTTAGAGGTCA; 3'- Sp17-inner: ATGCTATCGACATTGACCTAACTGACCCA; 3'RACE Adapter: GCGAGCACAGAATTAATATTTTTTTTTTTT; primers used for 5'RACE of the Sp17 gene: 5'-Sp17-outter: GCTCCGAACTGAATGA; 5'-Sp17-inner: TTTTGGCTGAGCCCGTAG; 6. The method for preparing the sperm surface protein of squid mansoni according to claim 2, characterized in that: the gene clone of the sperm surface protein Sp17 of squid mansoni comprises full-length cDNA splicing: the verified Sp17 The core sequence and the 3' and 5' end sequences were spliced to obtain the full-length cDNA sequence of the gene, and the physical and chemical properties of the cDNA were predicted according to the amino acid sequence of the cDNA and a phylogenetic tree was constructed. 7. The method for preparing the sperm surface protein of Squid mansoni according to claim 2, characterized in that: said tissue expression specificity analysis comprises extracting the total RNA of each tissue and reverse transcription: extracting sexually mature males respectively Total RNA from the brain, optic lobe, muscle, stomach, gill, liver, heart, testis, vas deferens, seminal vesicle, prostate, spermatophore, intestine and pancreas of squid mansoni was prepared by reverse transcription in real-time PCR laboratory The required cDNA template; before reverse transcription, the interference of genomic DNA in the extracted total RNA to the real-time PCR experiment needs to be removed. The specific steps are as follows: Add the following reaction system to the 200ml centrifuge tube: Total RNA 1ng 5×gDNA Eraser Buffer 2µl gDNA Eraser 1µl Water (Rnase ^- ) 8µl After mixing, centrifuge for a while, place the centrifuge tube in a PCR instrument, set the reaction conditions as follows: 42°C for 2 minutes, take it out quickly after the reaction and place it in an ice bath for 2 minutes. 8. The method for preparing the surface protein of Squid Mansoni according to claim 2, characterized in that: said tissue expression specificity analysis comprises real-time PCR primer design: according to the obtained Sp17 gene full-length cDNA sequence The primers designed for real-time PCR are: Sp17-YF: TCATTCAGTTCGGAGCCA; Sp17-YR: CCTATCTTCCAAATCAGCACA; Actin-F: TGAGAGGGAGATTGTGCGTG; Actin-R: GAACATAGATTCTGGAGCACGG. 9. the method for preparing the surface protein of Squid mansoni according to claim 2, is characterized in that: described tissue expression specificity analysis comprises fluorescence quantitative PCR: adopts the method for relative fluorescence quantification, selects Sp17 gene in the muscle The expression level was used as a reference, and the β-actin gene ( S.japonica JN564496.1) was used as an internal reference gene to compare the Sp17 gene in the brain, optic lobe, muscle, stomach, gill, liver, The expression levels in 14 different tissues including heart, testis, vas deferens, seminal vesicle, prostate, spermatozoa, intestine and pancreas were analyzed, and the significance of the difference was analyzed. 10. The use of the sperm surface protein of squid mansoni, which is characterized in that it is used for improving the reproductive regulation of squid mansoni and the use in cancer detection and treatment.