CN103705918B - Anti-porcine epidemic diarrhea virus hyperimmune serum and preparation method thereof - Google Patents

Abstract

The invention discloses that an inactivated vaccine of porcine epidemic diarrhea virus ZJ08 strain CGMCC No.7806 is used as an immunogen to immunize 50-60-day-old binary pigs, and hyperimmune serum is prepared through three times of immunization. The hyperimmune serum prepared by the invention has good properties, no bacteria, mold, mycoplasma and exogenous virus pollution, strong specificity, good safety, and the cure rate of artificial infection reaches 100%. Serum neutralization titer is above 2 ⁶ , the serum is subpackaged, stored at -70°C, and stored for 12 months.

Description

Anti-porcine epidemic diarrhea virus hyperimmune serum and preparation method thereof

technical field

The invention relates to a preparation method of antiviral serum, in particular to anti-porcine epidemic diarrhea virus hyperimmune serum and a preparation method thereof.

Background technique

Porcine epidemic diarrhea (PED) is an acute, contagious viral intestinal infectious disease that causes diarrhea in pigs, characterized by watery diarrhea, vomiting, dehydration, and decreased appetite. PEDV can persist in pig herds, and pigs of all ages are susceptible. The morbidity rate of suckling piglets, shelf pigs and fattening pigs can reach 100%, especially severe with suckling piglets, and the case fatality rate reaches 100%. The disease is a viral disease, and there is currently no effective treatment. When pregnant sows are vaccinated, piglets can acquire resistance through passive immunity through colostrum, but with the disappearance or unevenness of maternal antibodies, the resistance of piglets gradually decreases or is different, and the susceptibility to epidemic diarrhea It also behaves differently, and once the disease occurs, it will cause huge economic losses. The use of hyperimmune serum for group therapy is to enable piglets to acquire immunity in a passive immunization manner to achieve the purpose of protecting piglets. So far, this approach has worked. However, once the clinically popular strain of porcine epidemic diarrhea virus mutates, the therapeutic effect of the hyperimmune serum prepared therefrom will be weakened, and the morbidity and mortality of infected pigs will increase.

Contents of the invention

In order to solve the above problems, the present invention provides an anti-porcine epidemic diarrhea virus hyperimmune serum and a preparation method thereof.

Firstly, the present invention provides an anti-porcine epidemic diarrhea virus hyperimmune serum, which is obtained by separating the serum from immunized pigs with the inactivated vaccine of porcine epidemic diarrhea virus ZJ08 strain CGMCC No.7806 as the immunogen.

Wherein, the inactivated vaccine of porcine epidemic diarrhea virus ZJ08 strain CGMCC No.7806 is prepared by emulsifying porcine epidemic diarrhea virus ZJ08 strain and oil adjuvant.

The present invention also provides the preparation method of described anti-porcine epidemic diarrhea virus hyperimmune serum, comprising the steps of:

1) Immunization: Use the porcine epidemic diarrhea virus inactivated vaccine as the immunogen to immunize 50-60-day-old binary pigs, the first immunization: intramuscular injection of 4ml in total, the second immunization: in the first 14 days after immunization, the immunization dose is 8ml, injected at two points, the third immunization: 14 days after the second immunization, the immunized dose is 16ml, injected at three points;

2) Blood collection: 15 to 21 days after the last immunization, detect the neutralizing antibody titer, and when it reaches ²⁹ or more, kill the pig for aseptic bloodletting;

3) Serum separation and preparation: Cut the coagulated blood into small pieces of 1-3 square centimeters with a sterile knife, filter the serum, pack it into sterile saline bottles, and store the rest at -70°C.

The PEDV ZJ08 strain of the present invention is an attenuated strain of porcine epidemic diarrhea virus obtained by the applicant from a strong strain isolated from a pig farm in Zhejiang Province after 105 generations of attenuation. It is a current domestic PEDV strain. It is stable, safe, and has good immunogenicity. It can resist the attack of the current clinically popular strong virus strains, and the protection rate is over 90%.

The isolated PEDV 105-145 generation attenuated strain of PEDV was named Porcine Epidemic Diarrhea Virus ZJ08 strain, and it was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee; the preservation address is: No. 1 Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No. 7806, and the preservation time is June 28, 2013.

The invention successfully trial-produces hyperimmune serum against porcine epidemic diarrhea virus, which has good physical properties, no bacteria, mold and mycoplasma growth, no exogenous virus pollution, and the neutralizing antibody titer is more than ²⁶ times. The test results show that this serum not only has high antibody titer, but also has strong specificity, which fully meets the requirements of the serum standard for testing, and provides a powerful tool for evaluating the immune efficacy of vaccines. The serum potency evaluation results show that the hyperimmune serum prepared by the invention has a higher cure rate.

Description of drawings

Figure 1 is a picture of PEDV CPE (400×).

Fig. 2 is the result of PCR detection of S gene of PEDV isolate. In the figure, 1: sample; 2: negative control; M: DNAMarker DL2000.

Figure 3 is the result of PCR detection of M gene of PEDV isolate. In the figure, 1: sample; 2: negative control; M: DNAMarker DL2000.

Figure 4 shows the results of phylogenetic analysis of the M gene of PEDV isolates.

Figure 5 is the result of PCR detection of ORF3 gene of PEDV isolate. In the figure, 1: sample; 2: negative control; M: DNAMarker DL2000.

Figure 6 shows the alignment results of different generations of PEDV ORF3 sequences.

Figure 7 shows the results of immunofluorescence detection of the hyperimmune serum of the present invention and classical swine fever virus (CSFV). Among them, A is the cell well inoculated with PEDV hyperimmune serum and CSFV mixture; B is the cell well inoculated with cell maintenance solution.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

Example 1 Preparation of Porcine Epidemic Diarrhea Virus ZJ08 Strain Immunogen

Take an appropriate amount of porcine epidemic diarrhea-positive small intestine from a pig farm in Zhejiang, scrape the intestinal mucosa and contents, add PBS at a ratio of 1:5 (weight:volume), freeze and thaw repeatedly 3 times, centrifuge to take the supernatant, and filter it with a 0.22 μm membrane After filtration, trypsin with a final concentration of 20 μg/ml was added to the filtrate, and treated at 37° C. for 1.5 hours.

Inoculate Vero cells with a monolayer according to the conventional method, inoculate the virus at a ratio of 10%, absorb at 37°C for 1 hour, supplement the cell maintenance solution (containing 10 μg/ml trypsin), and culture in a 37°C incubator. At the 17th passage, there were obvious and stable changes in CPE, the cells shrank, the granules increased, and the piles were like bunches of grapes, and the cells were damaged and shed (Figure 1).

Design the detection primer pair according to the S gene: the forward primer is 5'-GGATACTTTGGCCTCTTGTG-3'; the reverse primer is 5'-CGCACTCGGATTACTCACAGC-3', pre-denaturation at 95°C for 5min, 95°C for 45s, 50°C for 1min, and 72°C for 2min After 32 cycles, elongate at 72 °C for 5 min. The amplified fragment of PEDV is 484bp. The results are shown in Figure 2.

According to the gene sequence of PEDV in GenBank, primers for amplifying the M gene sequence were designed:

PEDV-M-F: GTCTTACATGCGAATTGACC

PEDV-M-R: GGCATAGAGAGATAATGGCA

The size of the amplified M gene fragment is: 808 bp, the result is shown in Figure 3, and the sequence is shown in SEQ ID No.5.

The results showed that compared with classic reference strains such as British CV777 strain, Belgian Brl/87 strain, Korean KPD-9F strain and Japanese JMe2 strain, PEDV (strain ZJ08) did not belong to an evolutionary branch, and obvious mutations had occurred (Fig. 4), is a newly discovered popular strain in China.

The primers for amplifying the ORF3 sequence are:

ORF3-F: TCCTAGACTTCAACCTTACG

ORF3-R: GGTGACAAGTGAAGCACAGA

The size of the amplified ORF3 fragment is: 833 bp (Figure 5).

The porcine epidemic diarrhea virus was continuously passaged on Vero cells for 100 passages, and cloned and purified during this process, and then continued to passage on Vero cells for 145 passages. The 145th generation virus was detected by RT-PCR, and the amplified S gene fragment was 484bp. The ORF3 gene sequence of PEDV ZJ08 strain suddenly appeared 49 nucleotide deletions in the 105th generation, and maintained a stable deletion of 49 nucleotides in the 105th, 125th, and 145th generations (Figure 6). The sequence is shown in SEQ ID No. .6 shown.

According to the appendix of the current "Chinese Veterinary Pharmacopoeia", the 105th to 145th generation viruses were tested for sterility, mycoplasma and foreign viruses, and the results showed no bacteria, mold, mycoplasma and foreign virus contamination.

The 105~145 generation attenuated strain seed virus isolated and attenuated was named PEDV ZJ08 strain, and its microorganism preservation number was CGMCC No. 7806; the preservation time was June 28, 2013; preservation unit: China Microorganism Culture Preservation Management Committee Ordinary Microbiology Center; the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

The 125th generation of PEDV ZJ08 strain and cell maintenance solution were inoculated on Vero cells that had grown to a single layer at a ratio of 1:50, and cultured at 37°C. When 80% of the cells showed pathological changes, the virus was harvested and the virus price was determined. ( The virus content is 10 ⁷ TCID ₅₀ /ml), add 0.3% formaldehyde, place in a 37°C constant temperature incubator for 24 hours to inactivate, shake several times during this period, add 3 times the amount of oil adjuvant, and prepare an inactivated vaccine by emulsification. Store at 4°C for later use.

Example 2 Serum Preparation

1) Immunization program

The first immunization: a total of 4ml intramuscular injection.

The second immunization: 14 days after the first immunization, the immunization dose is 8ml, and the injection is divided into two points.

The third immunization: 14 days after the second immunization, the immunization dose is 16ml, and injected in three points.

2) blood collection

15-21 days after the last immunization, (the blood was collected once, the serum titer was measured by the neutralization test method, and then bled to death, with an interval of 3-4 days), the pigs were checked for health. No feed was given for the first 12 hours, but drinking water was not restricted. The butcher's knife and the skin where the knife enters are disinfected in advance, and the shallow basin containing blood is cleaned and then steam-sterilized. Sterile bloodletting.

3) Serum separation and preparation

Serum was separated by natural coagulation and pressurization method. Cut the coagulated blood into small pieces of 1 square centimeter with a sterilized knife, filter out the serum, and divide it into steam-sterilized 500-ml normal saline bottles, take 20ml out of each bottle to be tested, and store the rest at -70°C .

Porcine epidemic diarrhea virus ZJ08 strain was inactivated and emulsified with oil adjuvant to prepare immunogen. The neutralizing antibody titer was checked 15 days after the last immunization, and it reached ²⁶ or more. Afterwards, blood was collected aseptically and serum was separated according to conventional methods, and the purity of the semi-finished serum was tested. The results showed that there was no growth of bacteria, mold and mycoplasma in the prepared serum, and no exogenous virus contamination, all of which met the requirements.

Example 3 Determination of serum titer

The tested hyperimmune serum was diluted 1:2, 1:16, 1:32...1:512 with DMEM, and PEDV ZJ08 strain with an equal titer of 200 TCID ₅₀ /0.1ml was added to each dilution serum, 37 Neutralize at ℃ for 1 hour, inoculate 6 wells of a 96-well cell plate covered with a monolayer of Vero cells, 100 μl per well, and set up 6 wells of non-neutralizing virus positive control cells and 6 wells of negative control cells only inoculated with cell maintenance solution, 37 Cultivate in a 5% CO ₂ incubator for 3 to 5 days, and observe the CPE every day. The cells in the neutralization group and the negative control group should have no cytopathic changes, while the cells in the virus control group should have cytopathic changes. The highest dilution of serum that can protect 50% of the cells from pathological changes was taken as the neutralizing titer of the serum. It can be seen from Table 1 that the prepared anti-porcine epidemic diarrhea virus hyperimmune serum has a neutralizing titer above ²⁶ . This also provides a basis for how to use hyperimmune serum in the evaluation of vaccine efficacy in the future.

Table 1 The titer determination results of anti-PEDV hyperimmune serum

+: Represents cells with lesions; -: Represents cells without lesions

Example 4 Specificity Test

Dilute the serum to be tested by 2 times with DMEM nutrient solution, mix with 200TCID ₅₀ /0.1ml PEDV, TGEV, CSFV, PRV and PRRSV in equal amounts, and neutralize at 37°C for 1 hour, and inoculate the above-mentioned viruses with the neutralized mixture The proliferating cells were Vero, ST, PK, ST, Marc-145 cell monolayer in turn, 6 wells of 96-well cell plate, 100 μl per well, and 6 wells of non-neutralizing virus positive control cells and only inoculated with cell maintenance solution Negative control cells were cultured in 6 wells at 37°C in a 5% CO2 incubator for 3 to 5 days, and CPE was observed every day. There should be no cytopathic changes in the cell wells of the neutralization group and the negative control group, and the cells of the non-neutralization group and the virus control group Cytopathies should appear, and swine fever virus should be tested by immunofluorescence.

The results of the specificity experiment showed that the hyperimmune serum can only neutralize PEDV, and the neutralized mixture did not show cytopathic changes after inoculating the cells. After inoculating the cells with the mixture of several other sera and viruses, all except the swine fever group showed cell disease. The PK cells inoculated with the serum swine fever mixture showed fluorescence in each well by immunofluorescence detection (as shown in Figure 7). The above results show that the hyperimmune serum cannot neutralize TGEV, CSFV, PRV and PRRSV, and the specificity of the serum is very strong.

Example 5 Safety inspection

Five mice weighing 18-22 g were used, each subcutaneously injected with 0.5 ml of serum, observed for 10 days, and all should be healthy and alive. The results showed that: 10 days after the serum was inoculated into the mice, the mice were still alive without any clinical symptoms.

Embodiment 6 efficacy test

Thirty 14-day-old pigs from the same source without neutralizing antibodies to porcine epidemic diarrhea were divided into three groups: 10 pigs in the first group were given oral administration of 1000×MID/ml p55 strain of porcine epidemic diarrhea (provided by Fujian Academy of Agricultural Sciences) The 4th generation tissue is highly toxic) 1ml, inject 2ml of serum prepared by the present invention at the same time; The second group of 10, each oral administration of 1000×MID/ml porcine epidemic diarrhea virus p55 strain (Fujian Academy of Agricultural Sciences provides the 4th generation tissue At the same time, inject 2ml of hyperimmune serum prepared by PEDV attenuated vaccine strain CV777 (gifted by Harbin Veterinary Research Institute, which is made by weakening the strong virulence of Belgian PEDV) at the same time (serum titer and the present invention the same); the third group of 10 pigs was given 1ml of 1000×MID/ml porcine epidemic diarrhea virus p55 strain (the 4th generation tissue virulence provided by Fujian Academy of Agricultural Sciences) orally as a control. The control pigs should have typical diarrhea symptoms within 1 to 3 days after injection, and at least 8 died within 7 days. The first group and the second group of 10 pigs were observed for 7 days, and at least 80% of them were healthy and alive as qualified. The efficacy test results (Table 2), the control pigs had typical diarrhea symptoms within 3 days after injection, and all 10 pigs died within 7 days. The 10 pigs in the first group were all healthy and alive after 7 days of observation. The second group of pigs was observed for 7 days, and 3 pigs died, indicating that the hyperimmune serum prepared by the present invention has a better therapeutic effect on the clinically isolated PEDV p55 strain.

Table 2 The results of hyperimmune serum potency test

group Type of inoculum Test piglets observation time Number of cases cure rate 1 PEDV (V-ZJ08 strain) + serum of the present invention 10 7 0 100% 2 Serum prepared from PEDV (V-ZJ08 strain) + PEDV classic vaccine strain 10 7 3 70% 3 PEDV (V-ZJ08 strain) 10 7 10 -

Embodiment 7 shelf life test

Store the hyperimmune serum of porcine epidemic diarrhea virus ZJ08 strain at -70°C, and measure the serum titer at 1, 3, 6, 9, 12, and 15 months according to the method in Example 3.

The results showed that the serum neutralization titer was 2 ^6.9 when stored at -70°C for 15 months, and the serum storage period was set at -70°C for 12 months (see Table 3).

Table 3 The titer determination results of serum stored at -70°C for different time

different storage time 1 month 3 months 6 months 9 months 12 months 15 months serum neutralization titer 2^6.8 2^6.9 2^6.8 2^6.9 2^7.0 2^6.9

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

sequence listing

<110> Veterinary Medicine Research Center of Beijing Dabeinong Technology Group Co., Ltd.

Fuzhou Dabeinong Biotechnology Co., Ltd.

Beijing Kemufeng Biopharmaceutical Co., Ltd.

Beijing Dabeinong Technology Group Co., Ltd.

<120> Hyperimmune serum against porcine epidemic diarrhea virus and preparation method thereof

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Claims (3)

1. a kind of porcine epidemic diarrhea resisting virus hyper-immune serum, for ZJ08 plants of CGMCC of Porcine epidemic diarrhea virus The inactivated vaccine of No.7806 is immunogene, and serum is separated from immune swine and is made.

2. hyper-immune serum as described in claim 1, which is characterized in that described ZJ08 plants of CGMCC of Porcine epidemic diarrhea virus The inactivated vaccine of No.7806 is that ZJ08 plants of Porcine epidemic diarrhea virus inactivations and oil adjuvant emulsification are prepared.

3. the preparation method of hyper-immune serum of any of claims 1 or 2 comprising following steps:

1) it is immunized: with the inactivated vaccine of Porcine epidemic diarrhea virus ZJ08 plants of CGMCC No.7806 of any of claims 1 or 2 It grows up binary pig for immunogen immune 50-60 age in days, immune for the first time: the total 4ml of intramuscular injection, second immune: in first time It carries out within 14 days after immune, immunizing dose 8ml, a point two o'clock is injected, and third time is immune: carrying out within 14 days after immune at second, be immunized Dosage 16ml, point 3 points of injections；

2) it takes a blood sample: 15~21 days after last time is immune, detecting neutralize antibody titers, reach 2⁹When above, the sterile bloodletting of pig is killed；

3) serum separation and preparation: with the fritter for sterilizing knife solidification blood being divided into 1-3 square centimeters, serum is filtered out, nothing is sub-packed in In bacterium normal saline bottle, remaining is placed in -70 DEG C of preservations.