CN118086351A - Cryptosporidium parvum gene-deficient strain lacking 60kDa glycoprotein gene and its application - Google Patents

Abstract

The present invention discloses a Cryptosporidium parvum gene-deficient strain lacking a 60kDa glycoprotein gene and its application. The present invention provides a Cryptosporidium parvum GP60 gene-deficient strain and a construction method thereof, and successfully constructs a Cryptosporidium parvum gene-deficient strain by deleting the 60kDa glycoprotein GP60 gene through gene editing technology. Studies have shown that after the wild-type Cryptosporidium parvum IIdA20G1 subtype strain lacks the GP60 gene, it can still be cultured in vitro, and its in vitro growth load is significantly reduced, and the virulence of the gene-deficient strain is weakened, the load in mice is also significantly reduced, and the intensity of oocyst excretion is significantly weakened, and the death of GKO mice after infection will not be caused, and the virulence is significantly weakened, and it has the advantage of significantly weakened virulence, and can be used to prepare a Cryptosporidium parvum attenuated vaccine for preventing Cryptosporidium parvum infection.

Description

A gene-deficient strain of Cryptosporidium parvum lacking the 60kDa glycoprotein gene and its application

Technical Field

The present invention belongs to the field of genetic engineering technology, and more specifically, relates to a Cryptosporidium parvum gene-deficient strain lacking a 60kDa glycoprotein gene and its application.

Background technique

Cryptosporidium parvum is an important apicomplexan parasite, widely distributed in animals, humans and the environment. It is mainly transmitted through the fecal-oral route, parasitizing the intestinal epithelial cells of animals and humans. After infecting the host, it causes cryptosporidiosis with digestive disorders and watery diarrhea as the main clinical symptoms, and in severe cases causes the death of the host. The infection rate of Cryptosporidium is high, and the infection rate of young cattle and sheep can reach more than 70%. Because it can be transmitted across species to infect humans, it not only causes serious harm to the breeding industry, but also to public health.

Currently, the prevention and treatment methods for cryptosporidiosis are very limited, and only nitazoxanide has been approved by the FDA for clinical treatment of human cryptosporidiosis. Nitazoxanide has a therapeutic effect on cryptosporidiosis in adults with normal immunity and children over 1 year old, but the drug is ineffective for patients with AIDS and children with imperfect immune function development. In addition, there is no good vaccine for the prevention of cryptosporidiosis. Subunit vaccines such as peptides can only produce extremely low partial immune protection effects, and are still in the scientific research stage. There is currently no commercialized and effective Cryptosporidium vaccine on the market. At the same time, since Cryptosporidium is difficult to carry out continuous in vitro propagation and reverse genetic manipulation, its genetic manipulation technology has been in a backward state for a long time, resulting in insufficient understanding of the invasion and growth and development mechanism of Cryptosporidium. Therefore, no relevant vaccine candidate molecules have been studied and identified.

Cryptosporidium parvum mucin is a type of glycoprotein that exists specifically in Cryptosporidium and has a large number of glycosylation modifications. Among them, the glycoprotein with a molecular weight of 60kDa (GP60) may be involved in the adhesion process of Cryptosporidium and is very important for the invasion of Cryptosporidium; however, due to technical means such as gene knockout, its specific function is still unclear. Existing studies have shown that polyclonal antibodies used to recognize GP60 protein in vitro have the ability to block Cryptosporidium parvum from invading host cells through the study of GP60 recombinant protein and the preparation of its antibodies (Cui Zhaohui. Preliminary study on recombinant expression and related biological functions of Cryptosporidium parvum GP60 protein [D]. Henan Agricultural University, 2016.), but this study did not reveal the function of GP60 protein in Cryptosporidium parvum, and it is unknown whether it can be used as a vaccine target, and it is also unknown whether the relationship between GP60 protein and parasite virulence is known. Since there is currently a lack of vaccines or gene-deficient strains that can be used to prevent and treat Cryptosporidium, it is necessary to develop more gene-deficient strains or vaccines that can prevent Cryptosporidium infection, so as to provide more methods and technical support for the prevention and treatment of Cryptosporidium.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of existing gene-deficient strains or vaccines for preventing and treating Cryptosporidium infection, and to provide a gene-deficient strain of Cryptosporidium parvum lacking a 60kDa glycoprotein gene and an application thereof.

The first object of the present invention is to provide a method for constructing a Cryptosporidium parvum GP60 gene-deficient strain.

The second object of the present invention is to provide the application of the 60kDa glycoprotein gene of Cryptosporidium parvum.

The third object of the present invention is to provide a Cryptosporidium parvum GP60 gene-deficient strain.

The fourth objective of the present invention is to provide an application of a Cryptosporidium parvum GP60 gene-deficient strain.

The fifth object of the present invention is to provide a Cryptosporidium parvum vaccine.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention provides a method for constructing a Cryptosporidium parvum GP60 gene-deficient strain, wherein the CRISPR/Cas9 gene editing technology is used to knock out the Cryptosporidium parvum 60kDa glycoprotein GP60 gene, thereby obtaining the Cryptosporidium parvum GP60 gene-deficient strain; the nucleotide sequence of the 60kDa glycoprotein GP60 gene is shown in SEQ ID NO: 1.

The specific steps include:

S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as template and GP60 gene sgRNA primers, the dual sgRNA CRISPR plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602 was constructed;

S2. Using UPRT-mCh-Nluc-P2A-neo-UPRT plasmid and gDNA of wild-type Cryptosporidium parvum strain as templates, GP60 gene primers were used to construct homologous recombinant plasmid GP60-mCh-Nluc-P2A-neo-GP60;

S3. The dual sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into the wild-type Cryptosporidium parvum strain, and the Cryptosporidium parvum GP60 gene-deficient strain was obtained through drug screening and PCR identification.

The present invention uses CRISPR/Cas9 technology to directly knock out the 60kDa glycoprotein GP60 gene of Cryptosporidium parvum, and successfully obtains the GP60 gene-deficient strain; and for the first time, it is found that the GP60 gene is related to the virulence of the worm body, and the infection intensity and virulence of the GP60 gene-deficient strain in IFN-γ knockout (GKO) mice are significantly reduced. The present invention compares the in vitro growth of the labeled strain and the missing strain and the excretion intensity of the oocysts in the in vivo infection, and shows that under the in vitro growth condition, the GP60 gene-deficient strain grows slowly; under the in vivo growth condition, the load of the strain is significantly reduced, and the excretion intensity of the oocysts of the GKO mice is significantly reduced, and it does not cause the death of the infected mice, and can be used as a new vaccine candidate strain for preparing a Cryptosporidium parvum vaccine.

Preferably, the GP60 gene sgRNA primer sequences used in step S1 are shown in SEQ ID NOs: 2 to 9.

Preferably, the GP60 gene primer sequences used in step S2 are shown in SEQ ID NOs: 10-17.

Preferably, the wild-type Cryptosporidium parvum in step S3 is a Cryptosporidium parvum IIdA20G1 subtype strain.

As a preferred embodiment, the present invention provides a more specific method for constructing a Cryptosporidium parvum GP60 gene-deficient strain:

(1) The starting strain is a wild-type Cryptosporidium parvum IIdA20G2R1 subtype strain of the Cryptosporidium genus of the Cryptosporidium family of the order Cryptosporidium, which has a 60 kDa glycoprotein gene. The nucleotide sequence of the 60 kDa glycoprotein GP60 gene is shown in SEQ ID NO: 1;

(2) Construction of the dual sgRNA CRISPR plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602: Using the pAct::Cas9-GFP-U6::sgTK plasmid as a template, the upstream and downstream primers were used for amplification, and the sgRNA specific to the TK target was replaced with the sgRNA1 specific to the 60kDa glycoprotein target. The plasmid was connected using the principle of homologous recombination and then transformed into DH5α to obtain the pAct::Cas9-U6::sgGP601 plasmid; the pAct::Cas9-U6::sgGP602 plasmid was constructed in the same way. Then, the GP60 CRISPR1 plasmid was used as a template, and the plasmid backbone was amplified with primers; then, the GP60 CRISPR2 plasmid was used as a template to amplify the sgRNA fragment, and finally the plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602 was constructed;

(3) Construction of GP60-mCh-Nluc-P2A-neo-GP60 homologous recombination plasmid: The 5' homology arm and 3' homology arm of the 60kDa glycoprotein gene, the complete reading coding frame proAc-mCh-terAc sequence expressing the spontaneous red fluorescent protein mCherry, the drug screening gene Neo, and the luciferase value determination gene Nluc were connected. Specifically, the genome of the starting strain of Cryptosporidium parvum IIdA20G1 subtype strain was used as a template to design primers to amplify gp60-5'UTR and gp60-3'UTR, the proAc-mCh-terAc fragment was amplified from the plasmid carrying proAc-mCh-terAc, and the Neo and Nluc fragments were amplified from the plasmid carrying Neo and Nluc. The pUC19 vector was linearized by PCR amplification, and the multi-fragment ligase ExnaseTM MultiS was used to connect to construct the recombinant plasmid GP60-mCh-Nluc-P2A-neo-GP60;

(4) Obtaining the Cryptosporidium parvum gene-deficient strain Δgp60: The sporozoite suspension after excystation of the oocysts in step (1) is mixed with the pAct::Cas9-U6::sgGP601-U6::sgGP602 plasmid obtained in step (2) and the pAct::Cas9-U6::sgGP601-U6::sgGP602 plasmid obtained in step (3), and then the mixture is electro-transfected into a wild-type Cryptosporidium parvum strain to obtain the Cryptosporidium parvum gene-deficient strain Δgp60.

The invention provides application of a reagent for deleting or knocking out a 60kDa glycoprotein gene of Cryptosporidium parvum in constructing a GP60 gene-deleted worm strain or in preparing a Cryptosporidium parvum vaccine.

The invention provides the use of a 60kDa glycoprotein gene of Cryptosporidium parvum as a target in screening or preparing a drug for preventing Cryptosporidium parvum infection.

The present invention provides a Cryptosporidium parvum GP60 gene-deficient strain, which is constructed by the above method.

The invention also provides the use of the Cryptosporidium parvum GP60 gene-deficient strain in the preparation of Cryptosporidium parvum vaccine.

In addition, the present invention provides a Cryptosporidium parvum vaccine, comprising a Cryptosporidium parvum GP60 gene-deficient strain.

Preferably, the vaccine is an attenuated vaccine.

The present invention has the following beneficial effects:

The present invention provides a Cryptosporidium parvum GP60 gene-deficient strain and a construction method and application thereof, and successfully constructs a Cryptosporidium parvum gene-deficient strain by deleting the 60kDa glycoprotein GP60 gene through gene editing technology. The present invention first discovered that the GP60 gene is related to the virulence of the worm body, and the infection intensity and virulence of the GP60 gene-deficient strain in IFN-γ knockout (GKO) mice are significantly reduced, and the lack of GP60 affects the late stage of the asexual reproduction stage of the worm body, and seriously affects the virulence of Cryptosporidium parvum. Studies have shown that after the wild-type Cryptosporidium parvum IIdA20G1 subtype strain lacks the GP60 gene, it can still be cultured in vitro, and its in vitro growth load is significantly reduced, and the load in mice is also significantly reduced, and the excretion intensity of the oocysts is significantly weakened, and the death of GKO mice after infection will not be caused, and the virulence is significantly weakened, and it can be used to prepare a Cryptosporidium parvum attenuated vaccine for preventing Cryptosporidium parvum infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the construction principle of the Δgp60 insect strain.

FIG. 2 is the agarose gel electrophoresis detection result of PCR identification of Δgp60 strain.

FIG. 3 is the result of evaluating the in vitro growth of the Δgp60 strain using an in vitro culture experiment.

FIG. 4 is the result of evaluating the load of Δgp60 strain in mice by in vivo infection observation using scanning electron microscopy.

FIG5 is the result of evaluating the infection intensity of the Δgp60 strain in mice by using the amount of oocyst excretion of infected mice.

FIG. 6 is the result of evaluating the virulence of the Δgp60 strain using the survival rate of mice infected in vivo.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

The pACT1::Cas9-GFP-U6::sgTK plasmid and pINS1-3HA-Nluc-P2A-neo plasmid used in the examples are both from the laboratory of L. David Sibley at the University of Washington, USA.

Reference for typing and identification of wild-type Cryptosporidium parvum subtype IIdA20G1 strains: Li N, Zhao W, Song S, Ye H, Chu W, Guo Y, et al. Diarrhoea outbreak caused by coinfections of Cryptosporidium parvum subtype IIdA20G1 and rotavirus in pre-weaned dairycalves. Transbound Emerg Dis. 2022; 69: e1606-17.

Example 1 Construction of Cryptosporidium parvum Δgp60 strain

1. Starting strain

The starting strain used in this embodiment is a wild-type Cryptosporidium parvum IIdA20G1 subtype strain of the Cryptosporidium genus of the Cryptosporidium family of the order Cryptosporidium (collected from a dairy farm in Heilongjiang and obtained by typing and identification, and then subcultured and preserved in this laboratory to date). The strain has a 60kDa glycoprotein (GP60) gene, and the nucleotide sequence of the gene is shown in SEQ ID NO: 1.

2. Construction of CRISPR knockout plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602

(1) gRNA primer design

The GP60 gRNA sequence was designed using the EuPaGDT (http://grna.ctegd.uga.edu/) online tool: the gRNA1 sequence was 5'-GGCTGGGGCTGAGAATACAG, and the PAM sequence was CGG; the gRNA2 sequence was 5'-CTGCTTTTGGACTCAGATAC, and the PAM sequence was AGG. At the same time, the primers for constructing the double sgRNA plasmid were designed and synthesized. The specific primer information is shown in Table 1 below.

Table 1 Primers required for construction of dual sgRNA plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602

(2) Construction of single gRNA CRISPR plasmid

Using pACT1::Cas9-GFP-U6::sgTK plasmid as template, based on the principle of homologous recombination, the designed gRNA sequence was added to the plasmid linearized primer, the plasmid was linearized and amplified, and a single sgRNA CRISPR plasmid was constructed. The PCR reaction system and procedure are shown in Tables 2 and 3 below.

Table 2 PCR reaction system

Table 3 PCR reaction program

The PCR product was digested with restriction enzyme Dpn I (Dpn I, 1235S) from Takara to remove the plasmid template. The reaction system is shown in Table 4 below.

Table 4 Dpn I reaction system

Mix the reaction solution, centrifuge briefly, heat the PCR amplification enzyme at 70℃, and then digest at 37℃ for 20min. After completion, use the full-strength gold PCR product purification kit ( The digested PCR product was recovered using a PCR Purification Kit (EP101-01), and then the concentration was determined using a nucleic acid protein analyzer (NanoDrop 2000) from Thermo Fisher Scientific, and then connected using a single fragment connection kit (ClonExpress II One Step Cloning kit, C112-01) from Novozymes. The connection system is shown in Table 5 below.

Table 5 Single fragment ligation system

Where X represents the amount of vector plasmid used, specifically (0.02×number of vector bases) ng/vector recovery concentration. Mix the reaction solution, centrifuge briefly, connect at 37°C for 30 minutes, put it on ice immediately after the connection, and then transform it into DH5α competent cells to construct the plasmid. In this process, the CRISPR plasmid containing gRNA1 is named GP60 CRISPR1, and the CRISPR plasmid containing gRNA2 is named GP60 CRISPR2.

(3) Construction of dual gRNA CRISPR plasmid

Using the GP60 CRISPR1 plasmid as a template, the plasmid backbone was PCR amplified using primers GP60 CRISPR vector-F/R, and the PCR reaction conditions and reaction procedures were shown in Tables 2 and 3. Using the GP60 CRISPR2 plasmid as a template, the gRNA fragment was amplified using primers GP60 cassette-F/R, and the PCR reaction conditions and reaction procedures were the same as above. The PCR product was then detected by 0.8% agarose gel electrophoresis, and the two correctly identified fragments were connected using the ClonExpress II One Step Cloning kit (C112-01) of Novozymes to construct a plasmid. The construction method was the same as above, and the constructed plasmid was finally named: pAct::Cas9-U6::sgGP601-U6::sgGP602.

3. Construction of homologous repair plasmid GP60-mCh-Nluc-P2A-neo-GP60

(1) Extraction of Cryptosporidium parvum gDNA

The Cryptosporidium parvum oocyst samples were extracted according to the instructions of the DNA extraction kit (DNeasy Blood & Tissue Kit, 69504) of Qiagen, Germany to extract Cryptosporidium parvum gDNA.

(2) PCR amplification of target fragment

Using the Cryptosporidium parvum gDNA prepared above and the template plasmid UPRT-mCh-Nluc-P2A-neo-UPRT as templates (constructed by conventional methods in the art, with homology arms connected at both ends, a complete reading coding frame proAc-mCh-terAc sequence of red fluorescent protein mCherry connected in the middle, as well as a drug screening gene Neo and a luciferase value determination gene Nluc), the primers designed in Table 6 were used to amplify the corresponding target fragments, respectively, and the specific amplification system and procedure were the same as shown in Tables 2 and 3.

Table 6 Primers required for constructing homologous template plasmid GP60-mCh-Nluc-P2A-neo-GP60

(3) Recovery of target fragments

Subsequently, the PCR product was detected by 0.8% agarose gel electrophoresis, and the target fragment was recovered using the DNA gel recovery kit (FastPure Gel DNA Extraction Mini Kit, DC30101) of Nanjing Novezan Company in accordance with the kit operating instructions, and the concentration of the gel-recovered fragment was detected using a nucleic acid protein concentration meter (NanoDrop 2000).

(4) Construction of GP60-mCh-Nluc-P2A-neo-GP60 homologous template plasmid

According to the instructions of Nanjing ClonExpress MultiS One Step Cloning Kit (C113-01), the four prepared fragments were connected by homologous recombination. The multi-fragment connection reaction system was prepared as shown in Table 7 below.

Table 7 Single fragment ligation system

X in the table indicates the amount of vector plasmid used, specifically (0.02×number of vector bases) ng/vector recovery concentration. The reaction solution was mixed, centrifuged briefly, connected at 37°C for 30 minutes, and quickly placed on ice after the connection was completed. Then, it was transformed into DH5α competent cells, sent for sequencing after transformation, and the plasmid was extracted from the strain identified correctly by sequencing and stored at -20°C for future use. The plasmid GP60-mCh-Nluc-P2A-neo-GP60 was successfully constructed.

4. Construction of the Δgp60 gene-deficient strain of Cryptosporidium parvum

(1) Obtaining oocysts of Cryptosporidium parvum IIdA20G1 subtype strain

Place a magnetic stirrer and a fecal sample containing oocysts of the IIdA20G1 subtype of Cryptosporidium parvum in a blue-mouthed bottle, place the blue-mouthed bottle on a magnetic stirrer, and stir at 4°C for 3 hours. When there is no granular feces, sieve it, first through a 20-mesh sieve, then through a 60-mesh sieve. Centrifuge the sieved fecal liquid at 4000rpm for 10 minutes, discard the supernatant, resuspend it with 1L of pure water, and centrifuge it again at 4000rpm for 10 minutes. Discard the supernatant and resuspend it with 1L of pure water, and let it stand for 8 minutes to remove larger impurity particles.

After the sedimentation is completed, take the supernatant and continue to centrifuge and wash. After discarding the supernatant, resuspend it with 160mL pure water. Then pre-prepare 1:4 sucrose solution (65mL saturated sucrose solution + 255mL pure water) and 1:2 sucrose solution (110mL saturated sucrose solution + 220mL pure water). Prepare 16 50mL centrifuge tubes, add 20mL 1:4 sugar solution to each tube first, and then use a syringe to slowly add 20mL of 1:2 sugar solution from the bottom. During this period, obvious stratification can be seen. Then slowly add the suspension from the previous step to the top layer of the sucrose solution and centrifuge at 1000g for 25min. Then discard the upper 20mL waste liquid, and then draw 20mL of oocyst solution into a 250mL centrifuge bottle, add precooled pure water in a 1:1 ratio, centrifuge at 4000rpm for 10min, and finally resuspend with 5mL precooled pure water to obtain an oocyst suspension.

Finally, cesium chloride density gradient centrifugation was used, 1 mL of cesium chloride was added to a 1.5 mL low-absorption centrifuge tube, and 500 μL of oocyst suspension was gently added to the upper layer of cesium chloride with a pipette, and centrifuged at 13200 rpm for 3 min. After centrifugation, a white oocyst band was visible at the 1 mL scale. 700 μL was drawn from the oocyst band layer and added to a low-absorption 15 mL centrifuge tube, and then pre-cooled pure water was added at a ratio of 1:1, centrifuged at 10000 g for 10 min, the supernatant was discarded, and the washing was repeated 2 times. Finally, the oocysts were resuspended with 1 mL of pre-cooled PBS, and the three antibodies were added at a ratio of 1:100 after mixing, counted using a hemocytometer, and stored in a cold storage at 4 °C.

(2) Acquisition of fresh sporozoites of Cryptosporidium parvum IIdA20G1 subtype strain

Pipette 2.5×10 ⁷ fresh oocysts and place them on an ice box. Add 200μL Clorox sterilized water and 600μL PBS and place on ice for 10min. Centrifuge at 4℃13200rpm for 3min, discard the supernatant completely, and wash the oocysts 3 times with PBS. Then resuspend the treated oocysts with 400μL 1% BSA, add 400μL 1.5% bile sulfonate to a final concentration of 0.75%, and incubate in a 37℃ water bath for 60min. After the excystation is completed, centrifuge at 13200rpm for 3min and discard the supernatant. Then use 1mL room temperature PBS to resuspend the sporozoite suspension, centrifuge at 13200rpm for 3min and discard the supernatant. Repeat twice.

(3) Electroporation of sporozoites and oral infection of GKO mice

Using the 4D nuclear transfection kit (SF Cell Line 4D-NucleofectorTM X KitL, V4XC-2024) of Lonza, Germany, the sporozoites were resuspended in 80 μL of electroporation buffer (composed of 65.6 μL SF buffer and 14.4 μL S1 buffer). The specific configuration of the reaction solution is shown in Table 8 below, where the required amount of CRISPR knockout plasmid and homology repair plasmid is 50 μg, and the concentration needs to be adjusted to 5000 ng/μL before electroporation.

Table 8 100μL electroporation system

The mixed liquid was transferred to an electroporation cup and electroporated using the AMAXA 4D-Nucleofector system of Lonza, Germany, using the EH100 program. The electroporated sporozoites were diluted with 200 μL PBS and placed at room temperature.

(4) Intragastric infection of GKO mice

Three 3-5 week old GKO mice (IFN-γ knockout mice were purchased from the Institute of Medical Experimental Animals, Chinese Academy of Medical Sciences, and bred at the Experimental Animal Center of South China Agricultural University) were prepared in advance. Each mouse was gavaged with 200 μL of 8% NaHCO ₃ solution to neutralize gastric acid. After 5 minutes, each mouse was gavaged with 100 μL of PBS solution containing electroporated sporozoites. 24 hours after gavage, GKO mice were replaced with 16 g/L paromomycin sulfate solution instead of ordinary drinking water for in vivo drug screening.

(5) Detection of luciferase value

Subsequently, the luciferase activity in the feces of infected mice was detected to monitor the excretion of Cryptosporidium oocysts. A fresh mouse feces was collected 4, 9, and 14 days after infection, placed in a 1.5 mL centrifuge tube, and 8 to 10 3 mm glass beads and 0.8 mL fecal lysis solution were added to it. The mixture was shaken for 1 min and then centrifuged at 19000 g for 1 min. 50 μL of supernatant was added to the ELISA plate, and then 50 μL of luciferase substrate buffer and luciferase substrate (Promega, USA, Nano-Glo Luciferase kit, N1120) were mixed at a ratio of 25:1. The sample was then incubated at room temperature in the dark for 3 min. Finally, the Nluc value was measured using a multifunctional microplate reader (Berteng Instruments, USA). Once the mouse feces were positive for luciferase, the mouse feces needed to be collected every day to obtain more Cryptosporidium oocysts and obtain the purified gene-edited strain Δgp60 strain.

(6) Identification of Δgp60 strains

PCR1, PCR2 and PCR3 primers were designed and synthesized to identify the purified gene-edited insect strains. The specific primer sequences are shown in Table 9 below. The reaction system and procedure for PCR detection are the same as those shown in Tables 2 and 3.

Table 9 PCR identification primers for Δgp60 strains

Primer name Primer sequence (5'-3') GP60KOPCR1-F (SEQ ID NO: 18) ATTGATCTGCAGTAATGTCTTAATG GP60KOPCR1-R (SEQ ID NO: 19) CCAACTCATTCTGACTTCC GP60KOPCR2-F (SEQ ID NO: 20) CAACGTATCGCCTTCTATCGTC GP60KOPCR2-R (SEQ ID NO: 21) TGAGCAAGAAGAGACACTCG GP60KOPCR3-F (SEQ ID NO: 22) ATGAGATTGTCGCTCATTATCG GP60KOPCR3-R (SEQ ID NO: 23) ACACGAATAAGGCTGCAAAG

The schematic diagram of the construction principle of the Δgp60 strain is shown in Figure 1. In the test results, the presence of bands in PCR1 and PCR2 indicates that mCherry, drug screening fragment Neo, and luciferase fragment Nluc have been integrated into the gp60 site of Cryptosporidium parvum; PCR3 detects whether the gRNA targets the gp60 site of the Cryptosporidium parvum genome. If the wild-type Cryptosporidium parvum in the control group has a PCR3 band, but the experimental group has no PCR3 band, it means that the gp60 gene has been successfully knocked out. The specific test results are shown in Figure 2, indicating that the Δgp60 strain was successfully constructed.

Example 2 In vitro growth experiment of Cryptosporidium parvum Δgp60 strain

In a gene-edited insect strain infection sample, the luciferase measurement value is linearly related to the number of insects, and the measurement value increases with the increase in the number of insects. The proliferation time of Cryptosporidium after invading host cells is fixed, so by comparing the luciferase expression at different time points, the effect of gene editing on insect proliferation can be roughly inferred. If gene deletion has an effect on insect proliferation, it may be observed that the proliferation of the insect slows down at a specific time point (developmental stage related to the missing gene). This embodiment evaluates the growth of gene-edited insect strains through in vitro experiments.

(1) In vitro infection

The Δgp60 strain oocysts obtained in Example 1 were used to infect HCT-8 cells (purchased from the cell bank of the Chinese Academy of Sciences, CBP60030), and 2% FBS1640 complete medium was used for cell culture. Cryptosporidium was inoculated after the cell confluence reached more than 60%. On a 48-well cell culture plate, firstly, fresh oocysts were aspirated and placed on an ice box, 200 μL of Clorox disinfectant and 600 μL of PBS were added, and the plates were placed on ice for 10 min; then, the plates were centrifuged at 4°C and 13200 rpm for 3 min, the supernatant was completely discarded, and the oocysts were washed 3 times with PBS; finally, the oocysts were resuspended in 2% FBS1640 medium on an intercellular clean bench, the old HCT-8 cell medium was discarded, and the resuspended oocyst suspension was added to HCT-8 cells; when the confluence of HCT-8 cells reached more than 80%, 20,000 oocysts were infected in each well; at the same time, the wild-type Cryptosporidium parvum strain GP40-HA/GP15-Flag was used as a control (with a drug screening label and an Nluc fluorescent label).

(2) Luciferase assay

There were four replicate wells in each experimental group, and the luciferase value of each well was measured at 3, 12, 24, 36, and 48 h after infection. The luciferase determination method was the same as in Example 1. After the determination was completed, the data were statistically analyzed using Graph Pad 9.0.

The experimental results are shown in Figure 3. At 3h, the growth of the Δgp60 strain was no different from that of the GP40-HA/GP15-Flag strain; at 12h and 24h, there was no difference between the two, indicating that the lack of GP60 did not affect the formation of schizonts. However, at 36h and 48h, the growth of the load of the Δgp60 strain slowed down significantly, and there was a significant difference in the luciferase detection value with the GP40-HA/GP15-Flag strain (P = 0.0094 at 36h, P = 0.0006 at 48h), indicating that the lack of GP60 may affect the late stage of the asexual reproduction stage of the insect.

Example 3 In vivo infection experiment of Cryptosporidium parvum Δgp60 strain

In vivo infection experiment is an important method to evaluate the pathogenicity of Cryptosporidium parvum. This study evaluated it through the in vivo worm load, oocyst excretion intensity and mouse survival curve.

1. Test treatment

The experiment was conducted using 3-5 week old GKO mice, each mouse was housed in a single cage and the mean weight of each group was basically the same. A Δgp60 group was set up: each mouse was inoculated with 1000 fresh oocysts of the Δgp60 strain; a wild-type Cryptosporidium parvum control group was set up: the GP40-HA/GP15-Flag strain was used, and 1000 fresh oocysts were also inoculated; and a blank group was set up without oocysts. All mice were treated with paromomycin (16 g/L) during the experiment.

2. Assessment of the insect load in the body

At the peak of infection (9th day after infection), tissues at the junction of the ileum and cecum of GKO mice were collected for preparation of scanning electron microscopy samples, and the differences in the worm load in the control group and the group infected with the Δgp60 worm strain were compared.

(1) Sample preparation

Starve the mice one day in advance, remove the mouse feed and replace the bedding. On the day of sampling, the mice were killed by dislocating the neck and the hair of the mice was sprayed with 75% alcohol to moisten it. The abdominal cavity was quickly opened, the entire intestine was taken out and gently placed in pre-cooled PBS. Find the ileocecal end, cut the tissue 1 to 2 cm in front of the ileocecal end, and place it in new pre-cooled PBS. After cutting the ileum longitudinally, rinse it in clean PBS three times. Next, cut the ileum tissue into small pieces of 1 to ² mm3.

(2) Sample fixation

The ileum tissue was placed in 0.1% glutaraldehyde fixative (80 μL of 25% glutaraldehyde dissolved in 20 mL of 4% paraformaldehyde) precooled at 4°C and fixed at 4°C for 4 h.

(3) Sample dehydration

Treat with 10%, 20% and 30% ethanol solutions in sequence, dehydrate for 30 minutes each time, and then dehydrate with 50% ethanol for 1 hour, all steps are completed at 4°C. Then dehydrate with 70%, 80% and 90% ethanol aqueous solutions in sequence, each for 1 hour. Finally, dehydrate with anhydrous ethanol for 1 hour, repeat once. Then, infiltrate with anhydrous ethanol/LR-White mixture 1 (volume ratio 3:1) for 2 hours, anhydrous ethanol/LR-White mixture 2 (volume ratio 1:1) for 2 hours, and anhydrous ethanol/LR-White mixture 3 (volume ratio 1:3) for 2 hours, all of which are completed at -20°C.

(4) Sample embedding polymerization

First, infiltrate in LR-White resin at -20℃ overnight, then replace with new LR-White resin and continue infiltrating at -20℃ for 24h. Replace with new LR-White resin again and polymerize the sample under ultraviolet light (wavelength of 365nm) at -25℃ for 3-4 days. After the sample is polymerized, collect it in a sample bag and mark it with information such as preparation date, sample type and quantity, and finally store it in a constant temperature box at 25℃.

(5) Sample fixation

Add 2.5% glutaraldehyde fixative (1 mL of 25% glutaraldehyde dissolved in 9 mL of 4% paraformaldehyde) and fix the sample at 4°C. The maximum fixation time is two months. After fixation, rinse with PBS (0.1M, pH=7.4) 4 times, 20 minutes each time. Then use osmium acid (0.2M) to fix at room temperature for 1 hour. After fixation, rinse with deionized water 4 times, 20 minutes each time.

(6) Sample dehydration

Dehydrate with 30% and 50% ethanol for 10 min each, 70% ethanol for 3 h, 80% and 90% ethanol for 10 min each, and 100% ethanol for 10 min, repeat once. All the above operations were performed at room temperature.

(7) Sample critical point drying

The sample is carefully placed in a critical point dryer containing anhydrous ethanol to dry the sample, and then the sample is mounted on a stage, that is, the sample is fixed on the sample stage using conductive glue.

(8) Sample vacuum coating

A layer of gold was sprayed on the surface of the sample and the sample holder using a vacuum coating apparatus, and the coated sample was observed under a field emission scanning electron microscope (Verios 460, FEI Company, USA).

The results are shown in Figure 4. The amount of worms in the intestines of mice infected with the Δgp60 strain was significantly lower than that in the control group, indicating that the loss of GP60 is very important for the growth of Cryptosporidium in vivo.

2. Determination of oocyst excretion intensity

The oocyst excretion intensity was measured by the fresh feces luciferase value. Fresh feces were collected every two days starting from the second day after infection to measure the fresh feces luciferase value. The luciferase determination method was the same as in Example 1, and the oocyst excretion intensity change curve was plotted using Graph Pad 9.0 software.

The results are shown in Figure 5. From 2 to 8 days after infection, the amount of oocyst excretion of the Δgp60 strain increased slowly. During this period, the luciferase value per gram of feces was about 100 times that of the control group. At the peak of excretion (9 to 12 days after infection), the difference was reduced to 10 times. In general, the intensity of oocyst excretion of the Δgp60 strain was significantly lower than that of the control group.

3. Drawing of mouse survival curve

During the experiment, the time of death of mice was recorded and the survival curve of mice was drawn. The spirit and survival status of mice were recorded every day, and the survival rate was drawn into Kaplan using Graph Pad 9.0 software. Meier survival plot.

The results are shown in FIG6 . All mice in the control group died within 17 days after infection, while mice in the Δgp60 group were monitored until 30 days without death, with a survival rate of 100%. This indicates that the absence of GP60 seriously affects the virulence of Cryptosporidium parvum.

In summary, the present invention uses CRISPR/Cas9 technology to directly knock out the GP60 gene of Cryptosporidium parvum, successfully obtains the GP60 gene-deficient strain, and discovers for the first time that the GP60 gene is related to the virulence of the worm body, and the infection intensity and virulence of the GP60 gene-deficient strain in IFN-γ knockout (GKO) mice are significantly reduced. The present invention compares the in vitro growth of the labeled strain and the missing strain and the excretion intensity of the oocysts in the in vivo infection, and shows that under the in vitro growth condition, the GP60 gene-deficient strain grows slowly; under the in vivo growth condition, the Δgp60 strain can continuously infect the GKO mouse model, the load of the strain is significantly reduced, and the excretion intensity of the oocysts of the GKO mice is significantly reduced. The lack of GP60 seriously affects the virulence of Cryptosporidium parvum, and will not cause the death of infected mice, and can be used as a new vaccine candidate strain for the preparation of Cryptosporidium parvum vaccine.

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims (10)

1. A method for constructing a Cryptosporidium parvum GP60 gene-deficient strain, characterized in that the 60 kDa glycoprotein GP60 gene of Cryptosporidium parvum is knocked out using CRISPR/Cas9 gene editing technology to obtain a Cryptosporidium parvum GP60 gene-deficient strain; the nucleotide sequence of the 60 kDa glycoprotein GP60 gene is shown in SEQ ID NO: 1. 2. The construction method according to claim 1, characterized in that it specifically comprises the following steps: S1. Using pAct::Cas9-GFP-U6::sgTK plasmid as template and GP60 gene sgRNA primers, the double sgRNA CRISPR plasmid pAct::Cas9-U6::sgGP601-U6::sgGP602 was constructed; S2. Using UPRT-mCh-Nluc-P2A-neo-UPRT plasmid and gDNA of wild-type Cryptosporidium parvum strain as templates, GP60 gene primers were used to construct homologous recombinant plasmid GP60-mCh-Nluc-P2A-neo-GP60; S3. The dual sgRNA CRISPR plasmid and the homologous recombination plasmid were co-transfected into the wild-type Cryptosporidium parvum strain, and the Cryptosporidium parvum GP60 gene-deficient strain was obtained through drug screening and PCR identification. 3. The construction method according to claim 2, characterized in that the sgRNA primer sequences of the GP60 gene in step S1 are shown in SEQ ID NOs: 2 to 9. 4. The construction method according to claim 2, characterized in that the GP60 gene primer sequences in step S2 are shown in SEQ ID NOs: 10 to 17. 5. The construction method according to claim 2, characterized in that the wild-type Cryptosporidium parvum strain in step S3 is a Cryptosporidium parvum IIdA20G1 subtype strain. 6. Use of a reagent for deleting or knocking out the 60 kDa glycoprotein gene of Cryptosporidium parvum in constructing a GP60 gene-deficient strain or in preparing a Cryptosporidium parvum vaccine, characterized in that the nucleotide sequence of the 60 kDa glycoprotein gene of Cryptosporidium parvum is as shown in SEQ ID NO: 1. 7. Use of a 60 kDa glycoprotein gene of Cryptosporidium parvum as a target in screening or preparing a drug for preventing Cryptosporidium parvum infection, characterized in that the nucleotide sequence of the 60 kDa glycoprotein gene of Cryptosporidium parvum is as shown in SEQ ID NO: 1. 8. A Cryptosporidium parvum GP60 gene-deficient strain, characterized in that it is constructed by the method described in any one of claims 1 to 4. 9. Use of the Cryptosporidium parvum GP60 gene-deficient strain according to claim 8 in the preparation of a Cryptosporidium parvum vaccine. 10. A Cryptosporidium parvum vaccine, characterized by comprising the Cryptosporidium parvum GP60 gene-deficient strain according to claim 8.