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CN111228463A - Detection method and application of rhTSG-6 antiviral activity on chick embryo-H2N 9 subtype avian influenza virus hemagglutination system - Google Patents

Detection method and application of rhTSG-6 antiviral activity on chick embryo-H2N 9 subtype avian influenza virus hemagglutination system Download PDF

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CN111228463A
CN111228463A CN202010116906.5A CN202010116906A CN111228463A CN 111228463 A CN111228463 A CN 111228463A CN 202010116906 A CN202010116906 A CN 202010116906A CN 111228463 A CN111228463 A CN 111228463A
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王明丽
蒋敏之
吴博
何志远
周炜
夏兵兵
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Wuhu Tianming Biotechnology Co ltd
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Abstract

The invention provides a detection method and application of rhTSG-6 in the hemagglutination system of avian influenza virus subtype H2N9, the result of the method for observing and detecting the rhTSG-6 antiviral activity shows that the rhTSG-6 can inhibit the multiplication and replication of avian influenza virus subtype H2N9 in chick embryo allantois, and the hemagglutination phenomenon is reduced or no phenomenon occurs at all. By taking the rhTSG-6 protein titer detection method as a standard for evaluating results, the rhTSG-6 protein titer detection results measured in different batches can be more reliable and credible; the method has clear result and high repeatability and accuracy; the method verifies that the titer of the rhTSG-6 against the H2N9 subtype avian influenza virus is up to more than 100000.00U/mL. Fills up the blank of the related field.

Description

Detection method and application of rhTSG-6 antiviral activity on chick embryo-H2N 9 subtype avian influenza virus hemagglutination system
Technical Field
The invention belongs to the fields of microbiology and immunology, and particularly relates to a detection method and application of rhTSG-6 in an antiviral activity on a chick embryo-H2N 9 subtype avian influenza virus hemagglutination system.
Background
A great deal of research shows that TSG-6 is an anti-inflammatory factor with obvious effect, and in the inflammatory process of hepatitis, arthritis, retinitis, tissue injury, keloid and the like, TSG-6 can inhibit the expression of inflammatory factors TNF- α, IL-1 β, IL-6 and the like, has the functions of inhibiting neutrophil infiltration and the like, and has strong anti-inflammatory action.
Disclosure of Invention
The invention discloses a method for detecting the antiviral activity of rhTSG-6 on a chick embryo-H2N 9 subtype avian influenza virus hemagglutination system and application thereof, wherein H9N2 subtype avian influenza virus mainly infects host susceptible cells through hemagglutinin protein on the surface of the avian influenza virus; the hemagglutinin protein can bind to sialic acid receptors on the surfaces of vertebrate erythrocytes, so that agglutination between erythrocytes occurs, which is called Hemagglutination Assay (HA). When a certain amount of specific antibody is pretreated with virus, hemagglutinin protein on the surface of the virus is blocked by the specific antibody, and HA activity is lost, which is called Hemagglutination Inhibition (HI) phenomenon.
Based on the principle, the rhTSG-6 has the phenomenon of similar antiviral activity to a specific antibody on the hemagglutination system of the chick embryo-H2N 9 subtype avian influenza virus, namely, the replication and proliferation of the H9N2 subtype avian influenza virus in chick embryo allantois can be inhibited. The activity can be measured as the degree of inhibition (half inhibition or full inhibition) of hemagglutination of H9N2 subtype avian influenza virus by a certain amount of rhTSG-6, i.e., the reciprocal of the dilution that half of the chick embryos (50%) can still be protected from virus attack by the highest dilution of the rhTSG-6 test sample per ml to cause HI phenomenon is defined as rhTSG-6 unit (or titer), often expressed as unit (U). The result is observed by HI phenomenon, and the potency of the tested rhTSG-6 antiviral activity is calculated according to the Reed-Muench formula and is about 1.0 multiplied by 103U～1.0×105U/mL/mL; the data of the inhibited virus proliferation in the allantoic fluids of the chick embryos were statistically analyzed by Spss16.0, and the results are shown in FIG. 5. As can be seen from FIG. 5, the HA potency of each rhTSG-6 group is significantly decreased compared to the virus control group and the negative control group, and the HA potency is decreased with the decrease of the rhTSG-6 concentration, so that the rhTSG-6 is judged to have significant antiviral effect, and the low dose group, the medium dose group, the higher dose group and the high dose group all show very significant virus proliferation inhibition effect (p)<0.01). The method can accurately and objectively observe and detect the rhTSG-6 antiviral activity, and fills up the blank of related fields.
The technical scheme adopted by the invention is as follows:
the invention provides application of recombinant human tumor necrosis factor α induced protein 6 in a medicament for resisting H9N2 subtype avian influenza virus, rhTSG-6 can effectively inhibit the proliferation of the H9N2 subtype avian influenza virus in chick embryos through a chick embryo/H9N 2 subtype avian influenza virus hemagglutination system, and can obviously reduce the hemagglutination titer of the H9N2 virus in chick embryo allantoic fluid, thereby verifying that the rhTSG-6 has the antiviral effect.
The method also provides a hemagglutination inhibition system for detecting the antiviral activity of the recombinant human tumor necrosis factor α induced protein 6, wherein the hemagglutination inhibition system is a hemagglutination inhibition system of recombinant human tumor necrosis factor α induced protein 6/chick embryo/H9N 2 subtype avian influenza virus.
The invention also provides a method for detecting the antiviral activity of the recombinant human tumor necrosis factor α induced protein 6 based on the recombinant human tumor necrosis factor α induced protein 6/chick embryo/H9N 2 subtype avian influenza virus hemagglutination inhibition system, wherein 9-10 day old chick embryos are used as test vectors, and H9N2 subtype avian influenza virus is used as an attack virus.
Further, the method comprises the steps of:
(1) treating chicken embryos;
(2) diluting rhTSG-6 to different concentrations, respectively inoculating into allantoic cavity of chick embryo in step (1), and inoculating 100EID at the same position50H9N2 subtype avian influenza virus suspension is placed in each corresponding chick embryo allantoic cavity of each test group, and a negative control group, a virus control group and an rhTSG-6 positive serum control group are arranged;
(3) and (3) incubating the inoculated chicken embryos, collecting allantoic fluid of the chicken embryos after incubation for 72h, respectively determining the HA titer of the allantoic fluid in the chicken embryos, simultaneously performing a Hemagglutination Inhibition (HI) test by using rhTSG-6 positive serum, and recording and counting results.
Further, the step (1) specifically includes: taking SPF (specific pathogen free) chick embryos of 9-10 days old, irradiating the surfaces of the chick embryos by using an egg detection lamp in a dark room, observing the survival state of the chick embryos, and removing dead chick embryos; the line of the air cell where the blood vessels were less distributed was marked with a pencil or crayon and the injection point was marked approximately 0.3cm above the air cell line.
In the step (2), the concentration of the rhTSG-6 after dilution is respectively 105U/mL、104U/mL、103U/mL、102U/mL, the inoculum dose was 0.1 mL/embryo for each concentration.
In the step (2), the 100EID50The using amount of the H9N2 subtype avian influenza virus suspension is 0.1 mL/embryo.
In the step (2), the negative control group is prepared by inoculating sterile PBS buffer solution into chick embryos; the virus control group is a chicken embryo inoculated with 0.1 mL/embryo 100EID50H9N2 subtype avian influenza virus suspension.
In the step (3), the incubation temperature is 34-38 ℃.
The method for detecting the rhTSG-6 antiviral activity based on the chick embryo/H9N 2 subtype avian influenza virus hemagglutination system provided by the invention has the advantages that the phenomenon is obvious and easy to observe, and the error caused by subjective judgment can be reduced to the greatest extent; the rhTSG-6 can be detected by obvious antiviral activity, and the detected rhTSG-6 has high biological activity and strong sensitivity and can better reflect the reagent antiviral activity of the rhTSG-6.
The results of observation and detection of rhTSG-6 antiviral activity by the method provided by the invention show that rhTSG-6 can inhibit replication and proliferation of H2N9 subtype avian influenza virus in chick embryo allantois, and the hemagglutination phenomenon is reduced or no phenomenon occurs at all. The result is used as the standard of the evaluation result, so that the rhTSG-6 protein titer detection results measured in different batches are more reliable and credible. The method has the advantages of clear result, high repeatability and high accuracy. The method proves that the titer of the rhTSG-6 against the H2N9 subtype avian influenza virus is as high as about 1.0 multiplied by 103～1.0×105U/mL or more. Fills up the blank of the related field.
Drawings
FIG. 1 shows the result of identifying rhTSG-6 protein by Western Blot; wherein M is protein marker, Lane 1 is total protein after empty vector thallus is broken, Lane 2 is rhTSG-6 sample;
FIG. 2 is a flow chart of the operation of the method for detecting the antiviral activity of rhTSG-6 in example 1;
FIG. 3 is a flow chart of a specific micro HA assay for detecting rhTSG-6 antiviral activity based on chick embryo/H9N 2 subtype avian influenza virus hemagglutination system in example 2;
FIG. 4 is a graph showing the results of H9N2 HI titrations in allantoic fluid from different doses of rhTSG-6 chick embryos, wherein the allantoic fluid from test wells is diluted from well 1 to well 9, and a corresponding duplicate well is set for each dilution; well 10 is virus control; the 11 th hole is a positive control group (namely rhTSG-6 antiserum is added); the 12 th hole is the negative control group (no rhTSG-6) chick embryo allantoic fluid;
FIG. 5 shows the results of H9N2 HA titration of allantoic fluids inoculated into chick embryos after mixing rhTSG-6 and corresponding antiserum at equal amounts and incubating at 37 ℃ for 1H; wherein allantoic fluid in the test well is diluted from the 1 st well to the 6 th well, and a corresponding duplicate well is arranged for each dilution; 7 th to 8 th wells are virus control wells; the 9 th to 10 th wells are rhTSG-6 positive control wells; well 11 is a negative serum control (i.e., no rhTSG-6 antiserum); the 12 th hole is negative control chick embryo allantoic fluid;
FIG. 6 shows the results of comparison of HA titers of H9N2 subtype avian influenza virus in allantoic fluid of chick embryos of each group.
Detailed Description
The present invention will be described in detail with reference to examples.
The rhTSG-6 freeze-dried powder injection is self-prepared by the following method (batch number: 20180924) according to the reference, and the titer is 1.0 multiplied by 10 through the detection of chick embryo-H9N 2 subtype avian influenza virus series5U/mL; the subtype H9N2 avian influenza virus and the positive serum were gifted by the veterinary college of Yangzhou university.
The preparation method of the rhTSG-6 freeze-dried powder injection comprises the following steps:
(1) the recombinant human TSG-6 gene shown in SEQUENCE LISTING 400 <1 > reported in Genebank is subjected to codon optimization, and the recombinant human TSG-6 gene shown in SEQUENCE LISTING 400 <2 > is obtained by using a chemical synthesis method, wherein the Codon Adaptation Index (CAI) before optimization is 0.73, and the CAI after optimization is 0.97.
(2) Subcloning the recombinant human TSG-6 gene obtained in the step (1) into a pET-32a expression vector, converting the expression vector into BL21 escherichia coli, coating an LB plate containing ampicillin for overnight culture, selecting a single colony on the LB plate to perform PCR and BamH I and HindIII double enzyme digestion identification, and if the result is positive, indicating that the construction of the expression vector is successful, obtaining recombinant human TSG-6 recombinant bacteria; PCR amplification and double digestion products show a single band near 800bp by agarose gel electrophoresis No. 1 lane;
the PCR identification primer is:
F1：GGATCCTGGGGTTTTAAAGATGGC(BamH Ⅰ)；
R1：AAGCTTTTACAGATGACTAAAGCGAC(Hind Ⅲ)。
(3) selecting recombinant human TSG-6 recombinant bacteria, carrying out shake culture in an LB culture medium containing 100 mu g/mL ampicillin, carrying out amplification culture in the LB culture medium containing 100 mu g/mL ampicillin for 2-3 h, adding 1.0mM IPTG (isopropyl-beta-thiogalactoside) with final concentration when OD (OD) value is measured to be 0.6-0.8, carrying out induced expression at 30 ℃ for 5h, and collecting bacteria; through SDS-PAGE electrophoretic analysis, the mycoprotein after IPTG induced expression for 5 hours has a visible dominant expression band at the position of 45.0kD, and the expression amount reaches 60 percent; identified by Western blot, the mycoprotein after IPTG induced expression for 5h can generate specific reaction with rabbit anti-human TSG-6 polyclonal antibody, a specific band appears at about 45kD, and the specificity is high;
(4) resuspending the thallus collected in the step (3) by using 200mL of PBS, and ultrasonically breaking the bacterial precipitate at 4 ℃, wherein the ultrasonic conditions are as follows: power: 400W, working for 3S, performing ultrasonic treatment at intervals of 3S for 6min, and repeating for 3-4 times; centrifuging at 12000r/min for 20min to separate supernatant and precipitate, and subjecting the separated inclusion body precipitate to inclusion, washing, denaturation and renaturation to obtain renaturated product, i.e. crude product of recombinant human TSG-6 protein. The precipitate, the supernatant and the thallus are respectively taken and detected by SDS-PAGE electrophoresis. The recombinant protein is expressed as inclusion body through SDS-PAGE electrophoretic analysis.
The method for washing, denaturation and renaturation comprises the following steps:
① washing, namely, using a washing buffer solution (50mmol/L Tris, 100mmol/L NaCl, 2mol/L urea, 1mmol/LEDTA, 0.5 percent TritonX-100, pH8.0) to carry out heavy suspension on the inclusion bodies (10g) according to the wet weight-to-volume ratio of 1: 20, washing for 2h, centrifuging at 12000r/min for 20min, taking the precipitate, and repeating the washing once again;
② denaturation, weighing the washed precipitate wet weight (4.8g), resuspending the precipitate (240ml) in lysis buffer (50mmol/L Tris, 100mmol/L NaCl, 7mol/L guanidine hydrochloride, 0.1% β -mercaptoethanol, pH 8.45) at a wet weight to volume ratio of 1: 50, placing on a magnetic stirrer overnight, and fully dissolving;
③ dilution renaturation, preparing renaturation buffer solution (50mmol/L Tris, 100mmol/L NaCl, 1mmol/L GSH, 0.2mmol/L GSSG, pH8.0) for renaturation, taking the dissolved protein solution, centrifuging at 12000r/min for 20min, taking supernatant, adding renaturation buffer solution with the same volume (adding 250ml renaturation buffer solution), standing for 3h at 4 ℃, adding renaturation buffer solution to dilute to 4 times of the original volume (adding 500ml renaturation buffer solution), standing for 3h at 4 ℃, finally adding renaturation buffer solution to dilute to 5 times of the original volume (adding 250ml renaturation buffer solution), and standing for 3h at 4 ℃.
(5) Filtering the crude product of the recombinant human TSG-6 protein, passing the crude product through a His-tag affinity chromatographic column, performing gradient elution by using an Elutionbuffer (50mM Tris-Cl and 500mM imidazole, pH8.0), collecting the protein showing the ultraviolet absorption peak of the recombinant human TSG-6 protein, dialyzing the protein in a Tris-HCl buffer solution with the volume of 10 times at the temperature of 4 ℃ for more than 6 hours, removing high-concentration imidazole by dialysis twice, adjusting the pH to 5.0, passing a 1M NaCl eluent through an anion exchange chromatographic column to collect a flow-through liquid, namely a pure product of the recombinant human TSG-6 protein, and detecting the purity of the prepared target protein by SDS-PAGE, wherein the purity of the prepared target protein reaches more than 90%. Western Blot identification by specific monoclonal antibodies is shown in FIG. 1.
(6) Mixing the pure recombinant human TSG-6 protein obtained in the step (5) with a freeze-drying protective agent according to the equal volume of 1:1, and then freezing and drying to obtain the rhTSG-6 freeze-dried powder injection with the specification of 40 mu g/per unit (namely, the amount of rhTSG-6 complete antigen contained in each recombinant human TSG-6 protein standard is 40 mu g); the lyoprotectant is PBS mixed solution of glycerol, mannitol and sucrose, and the final concentrations of the three in 10mmol/L PBS buffer solution are 100mL/L of glycerol, 0.12g/mL of mannitol and 0.025g/mL of sucrose.
Example 1
The method for detecting the antiviral activity of the recombinant human tumor necrosis factor α induced protein 6 based on the recombinant human tumor necrosis factor α induced protein 6/chick embryo/H9N 2 subtype avian influenza virus hemagglutination system comprises the following steps:
(1) chick embryo treatment
The SPF grade 10-day-old chick embryos are placed in a dark room and are irradiated downwards from an air chamber by an egg candler, so that the blood vessels of the live chick embryos are clear, and the chick embryos can survive when moving autonomously in the chick embryos. If the blood vessels disappear and the chick embryos still have no moving signs after slight shaking for many times, the chick embryos are judged to be dead embryos and should be removed. And drawing an air chamber line on the live chick embryo by using a crayon, and marking an injection point about 0.3cm above the position where the air chamber line avoids the blood vessel. The surface of the chick embryo at the air chamber was then sterilized with iodophor and alcohol, and the injection site was punctured with a blood lancet.
(2) Separately testing the toxic reaction of rhTSG-6 to chick embryo
Ten times of incremental gradient dilution is carried out on rhTSG-6 from the concentration of the stock solution, and the dilution is respectively set to be divided into a high dose group, a higher dose group, a medium dose group and a low dose groupQuantity group; the potency of rhTSG-6 was: 105U/mL、104U/mL、103U/mL and 102U/mL. rhTSG-6 with different doses is respectively inoculated into allantoic cavities of chick embryos, each chick embryo is inoculated with 0.1ml, and each dilution is inoculated with 4 chick embryos. A negative control group injected with an equal volume of PBS was also set. After inoculation, the injection port was sealed with paraffin and placed in an incubator for 72 h. Every 12h, whether each chick embryo survives normally or not is observed.
If all chick embryos in the negative control group should normally survive, and the experimental group injected with rhTSG-6 normally survives as the negative control group, the rhTSG-6 under all the concentration of the dose group in the experiment is judged to have no toxic effect on the chick embryos.
(3) rhTSG-6 antiviral activity test operation method
The operation is required to be carried out in a class II biosafety cabinet. The specific operation flow is shown in detail in fig. 2.
Setting rhTSG-6 into a high dose group, a medium dose group and a low dose group respectively, and diluting the rhTSG-6 to different concentrations by ten-fold increasing gradient; the potency of rhTSG-6 was: 105U/mL、104U/mL、103U/mL and 102U/mL. The diluted rhTSG-6 with different concentrations is inoculated into allantoic cavity of chick embryo at dosage of 0.1mL per embryo, and then 100EID is injected at the same position at dosage of 0.1mL per embryo50H9N2 subtype avian influenza virus suspension. Incubate at 37 ℃ for 72 h. And simultaneously setting a negative control group injected with PBS with the same volume, a virus control group only injected with virus suspension with the same volume and a positive control group injected with mixed liquor with the same volume of rhTSG-6 antiserum diluted at a ratio of 1:640 and rhTSG-6 antiserum diluted at a ratio of 1: 512.
After the pinhole of each group of inoculated chick embryos is sealed conventionally, the chick embryos are incubated at 37 ℃. After the chick embryos are inoculated, the chick embryos are observed 2 times in an egg examination room every day.
The embryos incubated for 72 hours were placed in a dark room, observed by shining an egg light down from the air chamber, and the survival of each group of embryos was recorded, followed by harvesting of allantoic fluid from each embryo for hemagglutination. First, a "negative control group" and a "virus control group" were detected; the abnormal change of the hemagglutination of the chick embryo allantoic fluid caused by no virus is indicated by "-", and the abnormal change of the hemagglutination of the chick embryo allantoic fluid caused by virus is indicated by "+". When the virus-induced chick embryo allantoic fluid hemagglutination phenomenon "+++ or ++++" appears in the virus control group, that is, all chick embryos in the virus control group are infected, according to the test result in the laboratory, the titer of the chick embryo allantoic fluid hemagglutination phenomenon is not lower than 1: 1000, when all the chick embryos in the negative control group grow well and the allantoic fluid has no hemagglutination phenomenon, the test shows that the control system is qualified, and the result judgment can be carried out.
Allantoic fluid is not clotting; this is the negative control result.
The result of the hemagglutination of the allantoic fluid is more than 75% of that of the allantoic fluid in the virus control group.
The result of the detection of the allantoic fluid in each experimental group of rhTSG-6 is judged as follows:
+ the allantoic fluid has hemagglutination, and the average titer is less than 25% of that of the chick embryo allantoic fluid in the virus inhibition control group;
the allantoic fluid has hemagglutination, and the average titer is less than 50% of that of the allantoic fluid of the chick embryo in the virus-inhibiting control group;
the allantoic fluid has hemagglutination phenomenon, and the average titer is less than 75% of that of the allantoic fluid in the virus inhibition control group;
the titer of the ++++ allantoic fluid hemagglutination is more than 75% of that of the allantoic fluid of the virus control group.
The rhTSG-6 titer determination takes the highest dilution capable of inhibiting the hemagglutination phenomenon of 50% chick embryo allantoic fluid as 1 rhTSG-6 unit, namely the highest dilution of each milliliter of rhTSG-6 test sample can still protect 50% chick embryos from virus attack, the reciprocal of the dilution is determined as 1 rhTSG-6 unit (or titer), and the calculation is carried out according to the Reed-Muench formula. By a Reed-Muench method, the rhTSG-6 titer in each test sample can be calculated by the HI phenomenon degree of the H9N2 subtype avian influenza virus infection chicken embryo in each test group.
(4) Detection of Hemagglutination Inhibition (HI) phenomena in allantoic fluid of chick embryos of each test group
50. mu.L of Phosphate Buffered Saline (PBS) was added dropwise to each well of the 96-well V-shaped microagglutinating plate in 2 rows. Diluting allantoic fluid of each group of chick embryo at a ratio of 1: 5, adding 50 μ L per well to the 1 st row of wells, diluting allantoic fluid of the test well from the 1 st well to the 9 th well, and setting a corresponding compound well for each dilution degree; the 10 th hole is H9N2 subtype avian influenza virus control; the 11 th hole is a positive control (namely rhTSG-6 antiserum is added) of a specific antibody; the 12 th well is a negative chick embryo allantoic fluid control. Adding 50 μ L of 0.5% chicken red blood cell suspension into each well, shaking slightly and mixing well, standing at room temperature for 40min, and observing the result. The maximal dilution hemagglutination inhibition potency of the rhTSG-6 group is determined by the complete inhibition (red dot shape) of the chicken erythrocyte agglutination. The results are shown in detail in FIG. 4.
Data on the inhibited virus proliferation in allantoic fluid from each group of chick embryos was recorded and statistically analyzed by Spss16.0, and the results are shown in FIG. 6. As can be seen from FIG. 6, compared with the virus control group and the negative control group, the HA titer of each rhTSG-6 group is obviously reduced, and the HA titer reduction amplitude is reduced along with the reduction of the rhTSG-6 concentration, so that the rhTSG-6 is judged to have obvious antiviral effect, and meanwhile, the low-dose group, the medium-dose group, the higher-dose group and the high-dose group all show very obvious virus proliferation inhibition efficacy (p < 0.01).
Example 2
Specific trace HA test for detecting rhTSG-6 antiviral activity based on chick embryo/H9N 2 subtype avian influenza virus hemagglutination system
The principle of the method is based on that rhTSG-6 has antiviral activity, but if a certain amount of rhTSG-6 recombinant protein and rhTSG-6 antiserum are added together firstly, mixed uniformly and put into a 37 ℃ incubator to act for 1H, and then inoculated into chick embryos of various test groups, the H9N2 subtype avian influenza virus in the chick embryos is not influenced in proliferation. The chick embryo allantoic fluid of the visible test group is detected to have obvious Hemagglutination (HA) phenomenon by a chick embryo/H9N 2 subtype avian influenza virus hemagglutination system. The assay is highly specific.
The preparation method of the rhTSG-6 antiserum comprises the following steps:
(1) selection of immunized animals: selecting 10 healthy and healthy non-infectious diseases of 5-month-old coming air cocks with the weight of 2-2.5 kg/cock, and breeding in cages by an animal center of Anhui medical university [ SYXK (Anhui) 2017-.
(2) A method of immunizing an animal comprising: the rhTSG-6 antiserum is prepared by diluting the protein concentration of the rhTSG-6 freeze-dried powder injection to 2.0mg/mL by using 0.01mol/L phosphate buffer solution (pH 7.0) to immunize a coming cock. The first immunization adopts Freund complete adjuvant to immunize for 1 time, then adopts Freund incomplete adjuvant to immunize for 2 times, and then adopts non-adjuvant to immunize for 2 times. Specifically, intramuscular injection is performed for 1 time per week, 1mL is performed for each time, after 5w of immunization, blood is drawn from the heart, and serum is centrifuged and separated.
(3) The titer of the obtained specific antiserum by an immune double diffusion method is not less than 1: 16. The antiserum is subjected to coarse purification by a saturated ammonium sulfate method and fine purification by an affinity chromatography method to obtain refined rhTSG-6 antiserum, and sterile filtration is carried out, then sterile glycerol with the volume of 50% is added, and the mixture is kept at-20 ℃ for later use.
The specific trace HA test for detecting the rhTSG-6 antiviral activity based on the chick embryo/H9N 2 subtype avian influenza virus hemagglutination system specifically comprises the following steps:
(1) preparing a mixed solution of the rhTSG-6 recombinant protein and the corresponding antiserum: diluting rhTSG-6 suspension at a ratio of 1:640, mixing with diluted rhTSG-6 antiserum at a ratio of 1: 512, placing in 37 deg.C incubator for 1h, and collecting antiserum mixture.
(2) Inoculating chick embryos: the antiserum mixture was inoculated into chick embryos at a dose of 0.1mL per embryo, followed by injection of 100EID at a dose of 0.1mL per embryo50H9N2 subtype avian influenza virus suspension. Incubate at 37 ℃ for 72 h. Meanwhile, a negative control group injected with PBS of the same volume only, a virus control group injected with virus suspension of the same volume only, an rhTSG-6 positive control group and a negative serum control group (namely, no rhTSG-6 antiserum) are arranged.
(3) And (3) observing chicken embryos: after the pinhole of each group of inoculated chick embryos is sealed conventionally, the chick embryos are incubated at 37 ℃. After the chick embryos are inoculated, the chick embryos are observed 2 times in an egg examination room every day. Chick embryo survival was observed after 72h in the dark and chick embryo allantoic fluid was harvested.
(4) Detection test of allantoic fluid: 50. mu.L of Phosphate Buffered Saline (PBS) was added dropwise to each well of the 96-well V-shaped microagglutinating plate in 2 rows. Diluting allantoic fluid in a test hole from a 1 st hole to a 6 th hole, and setting a corresponding multiple hole for each dilution degree; 7 th to 8 th wells are virus control wells; the 9 th to 10 th wells are rhTSG-6 positive control wells; well 11 is a negative serum control (i.e., no rhTSG-6 antiserum); allantoic fluid from chick embryos was negative in well 12. Adding 50 μ L of 0.5% chicken red blood cell suspension into each well, shaking slightly and mixing well, standing at room temperature for 40min, and observing the result.
(5) Interpretation of the results: when the chicken red blood cells in the virus control wells are completely agglutinated, the titer of the antiserum is judged, namely the phenomenon that the chicken red blood cells are completely agglutinated is taken as the titer of the maximum dilution of the antiserum. The results are shown in detail in FIG. 5.
As can be seen from FIG. 5, the results of the virus control group and the positive and negative control groups showed that the rhTSG-6 antiserum in the allantoic fluid of each test group interacted with the same amount of rhTSG-6, and the antiviral effect of the corresponding rhTSG-6 was completely lost. It was confirmed that this phenomenon has high specificity.
The above detailed description of the method and use of rhTSG-6 for detecting its antiviral activity in the hemagglutination system of avian influenza virus subtype avian H2N9 with reference to the examples is illustrative and not restrictive, and several examples can be cited within the scope of the present invention, therefore variations and modifications thereof without departing from the general inventive concept shall fall within the scope of the present invention.
SEQUENCE LISTING
<110> Wu lake Tianming Biotechnology Limited
<120> detection method and application of rhTSG-6 in antiviral activity of chick embryo-H2N 9 subtype avian influenza virus hemagglutination system
<130>1
<160>3
<170>PatentIn version 3.3
<210>1
<211>780
<212>DNA
<213> TSG-6 Gene before optimization
<400>1
tggggattca aggatggaat ttttcataac tccatatggc ttgaacgagc agccggtgtg 60
taccacagag aagcacggtc tggcaaatac aagctcacct acgcagaagc taaggcggtg 120
tgtgaatttg aaggcggcca tctcgcaact tacaagcagc tagaggcagc cagaaaaatt 180
ggatttcatg tctgtgctgc tggatggatg gctaagggca gagttggata ccccattgtg 240
aagccagggc ccaactgtgg atttggaaaa actggcatta ttgattatgg aatccgtctc 300
aataggagtg aaagatggga tgcctattgc tacaacccac acgcaaagga gtgtggtggc 360
gtctttacag atccaaagca aatttttaaa tctccaggct tcccaaatga gtacgaagat 420
aaccaaatct gctactggca cattagactc aagtatggtc agcgtattca cctgagtttt 480
ttagattttg accttgaaga tgacccaggt tgcttggctg attatgttga aatatatgac 540
agttacgatg atgtccatgg ctttgtggga agatactgtg gagatgagct tccagatgac 600
atcatcagta caggaaatgt catgaccttg aagtttctaa gtgatgcttc agtgacagct 660
ggaggtttcc aaatcaaata tgttgcaatg gatcctgtat ccaaatccag tcaaggaaaa 720
aatacaagta ctacttctac tggaaataaa aactttttag ctggaagatt tagccactta 780
<210>2
<211>783
<212>DNA
<213> optimized TSG-6 Gene
<400>2
tggggtttta aagatggcat ttttcataat agcatctggc tggaacgcgc cgccggcgtt 60
tatcatcgtg aagcccgtag tggcaaatat aaactgacct atgcagaagc caaagcagtg 120
tgcgaatttg aaggcggtca tctggcaacc tataaacagc tggaagcagc ccgtaaaatt 180
ggctttcatg tgtgcgcagc cggttggatg gccaaaggtc gcgtgggcta tccgattgtg 240
aaaccgggcc cgaattgcgg ttttggcaaa accggtatta ttgattatgg tattcgtctg300
aatcgtagtg aacgttggga tgcctattgt tataatccgc atgcaaaaga atgcggtggc 360
gtttttaccg atccgaaaca gatttttaaa agcccgggct ttccgaatga atatgaagat 420
aatcagatct gctactggca tattcgtctg aaatatggtc agcgcattca tctgagtttt 480
ctggattttg atctggaaga tgatccgggc tgcctggcag attatgttga aatctatgat 540
agctatgacg atgttcatgg ttttgtgggt cgctattgtg gtgacgaact gccggatgat 600
attattagta ccggcaatgt gatgaccctg aaatttctga gtgatgccag cgtgaccgca 660
ggcggttttc agattaagta tgttgcaatg gaccctgtga gcaaaagcag ccagggcaaa 720
aataccagta ccaccagtac cggtaataag aattttctgg ccggtcgctt tagtcatctg 780
taa 783
<210>3
<211>260
<212>PRT
<213> amino acid sequence of recombinant human TSG-6
<400>3
Trp Gly Phe Lys Asp Gly Ile Phe His Asn Ser Ile Trp Leu Glu Arg
1 5 10 15
Ala Ala Gly Val Tyr His Arg Glu Ala Arg Ser Gly Lys Tyr Lys Leu
20 25 30
Thr Tyr Ala Glu Ala Lys Ala Val Cys Glu Phe Glu Gly Gly His Leu
35 40 45
Ala Thr Tyr Lys Gln Leu Glu Ala Ala Arg Lys Ile Gly Phe His Val
50 55 60
Cys Ala Ala Gly Trp Met Ala Lys Gly Arg Val Gly Tyr Pro Ile Val
65 70 75 80
Lys Pro Gly Pro Asn Cys Gly Phe Gly Lys Thr Gly Ile Ile Asp Tyr
85 90 95
Gly Ile Arg Leu Asn Arg Ser Glu Arg Trp Asp Ala Tyr Cys Tyr Asn
100 105 110
Pro His Ala Lys Glu Cys Gly Gly Val Phe Thr Asp Pro Lys Gln Ile
115 120 125
Phe Lys Ser Pro Gly Phe Pro Asn Glu Tyr Glu Asp Asn Gln Ile Cys
130 135 140
Tyr Trp His Ile Arg Leu Lys Tyr Gly Gln Arg Ile His Leu Ser Phe
145 150 155 160
Leu Asp Phe Asp Leu Glu Asp Asp Pro Gly Cys Leu Ala Asp Tyr Val
165 170 175
Glu Ile Tyr Asp Ser Tyr Asp Asp Val His Gly Phe Val Gly Arg Tyr
180 185 190
Cys Gly Asp Glu Leu Pro Asp Asp Ile Ile Ser Thr Gly Asn Val Met
195 200 205
Thr Leu Lys Phe Leu Ser Asp Ala Ser Val Thr Ala Gly Gly Phe Gln
210 215220
Ile Lys Tyr Val Ala Met Asp Pro Ala Ser Lys Ser Ser Gln Gly Lys
225 230 235 240
Asn Thr Ser Thr Thr Ser Thr Gly Asn Tyr Asn Phe Leu Ala Gly Arg
245 250 255
Phe Ser His Leu
260

Claims (7)

1. The recombinant human tumor necrosis factor α induced protein 6 is applied in medicine for resisting H9N2 subtype avian influenza virus.
2. A hemagglutination inhibition system for detecting the antiviral activity of recombinant human tumor necrosis factor α -induced protein 6 is characterized in that the hemagglutination inhibition system is a hemagglutination inhibition system of recombinant human tumor necrosis factor α -induced protein 6/chick embryo/H9N 2 subtype avian influenza virus.
3. The method for detecting the antiviral activity of the recombinant human tumor necrosis factor α induced protein 6 based on the recombinant human tumor necrosis factor α induced protein 6/chick embryo/H9N 2 subtype avian influenza virus hemagglutination inhibition system is characterized in that 9-10 day-old chick embryos are used as test vectors, and H9N2 subtype avian influenza virus is used as an attack virus.
4. A method according to claim 3, characterized in that the method comprises the steps of:
(1) treating chicken embryos;
(2) diluting rhTSG-6 to different concentrations, respectively inoculating into allantoic cavity of chick embryo in step (1), and inoculating 100EID at the same position50H9N2 subtype avian influenza virus suspension is placed in each corresponding chick embryo allantoic cavity of each test group, and a negative control group, a virus control group and an rhTSG-6 positive serum control group are arranged;
(3) and (3) incubating the inoculated chicken embryos, collecting allantoic fluid of the chicken embryos after incubation for 72h, respectively determining the HA titer of the allantoic fluid in the chicken embryos, simultaneously performing a Hemagglutination Inhibition (HI) test by using rhTSG-6 positive serum, and recording and counting results.
5. The method according to claim 3, wherein the rhTSG-6 is diluted to a concentration of 10 in step (2)5U/mL、104U/mL、103U/mL、102U/mL, the inoculum dose was 0.1 mL/embryo for each concentration.
6. The method of claim 3, wherein in step (2), the 100EID is performed by50The using amount of the H9N2 subtype avian influenza virus suspension is 0.1 mL/embryo.
7. The method according to claim 3, wherein in the step (3), the incubation temperature is 34-38 ℃.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
US6313091B1 (en) * 1995-07-20 2001-11-06 New York University Pharmaceutical compositions containing TSG-6 for treating inflammatory diseases and cancer-related pathologies and method
JP2012500792A (en) * 2008-08-22 2012-01-12 アンスロジェネシス コーポレーション Methods and compositions for the treatment of bone defects with placental cell populations
US20160075750A1 (en) * 2011-11-04 2016-03-17 Darwin J. Prockop Production of TSG-6 Protein
US20160184330A1 (en) * 2014-12-31 2016-06-30 Signpath Pharma Inc. Suppression of Cytokine Release and Cytokine Storm

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6313091B1 (en) * 1995-07-20 2001-11-06 New York University Pharmaceutical compositions containing TSG-6 for treating inflammatory diseases and cancer-related pathologies and method
JP2012500792A (en) * 2008-08-22 2012-01-12 アンスロジェネシス コーポレーション Methods and compositions for the treatment of bone defects with placental cell populations
US20160075750A1 (en) * 2011-11-04 2016-03-17 Darwin J. Prockop Production of TSG-6 Protein
US20160184330A1 (en) * 2014-12-31 2016-06-30 Signpath Pharma Inc. Suppression of Cytokine Release and Cytokine Storm

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赵增成等: "银翘散抗禽流感病毒体外抑制试验", 《山东农业科学》 *
赵雨等: "重组鸡α干扰素抑制H9N2禽流感病毒在鸡胚中增殖的研究", 《中国预防兽医学报》 *

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