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CN-121975743-A - Porcine circovirus 2-type haemophilus parasuis disease bivalent inactivated vaccine and preparation method thereof

CN121975743ACN 121975743 ACN121975743 ACN 121975743ACN-121975743-A

Abstract

The invention discloses a porcine circovirus type 2 and haemophilus parasuis disease combined inactivated vaccine and a preparation method thereof. The invention respectively separates and obtains the porcine circovirus strain, the haemophilus parasuis serum 4 strain and the haemophilus parasuis serum 5 strain which are popular in the field, the microorganism preservation numbers of the porcine circovirus strain, the haemophilus parasuis serum 4 strain and the haemophilus parasuis serum 5 strain are CGMCC No.47094, CGMCC No.46698 and CGMCC No.46699, the separated strains or strains are prepared into the bivalent inactivated vaccine, lentinan is added in the vaccine preparation process to enhance the immune effect, the safety evaluation result shows that no local or systemic adverse reaction caused by the bivalent vaccine occurs, the safety is good, and the immune effect evaluation result of the bivalent vaccine shows that the bivalent vaccine can provide complete protection for animals aiming at the porcine circovirus, the haemophilus parasuis serum 4 strain and the haemophilus parasuis serum 5 which are popular in the field and has good immune protection effect.

Inventors

  • ZHANG ZHIMING
  • CHAI ZHIYUAN
  • CHAI HUA
  • LIU HONGBIN
  • LIANG WANNAN
  • LI YINGMING
  • FANG CHAO
  • LI JIANHUA
  • WANG HAO
  • HU RUIHONG

Assignees

  • 哈药集团生物疫苗有限公司

Dates

Publication Date
20260505
Application Date
20251229

Claims (10)

  1. 1. The porcine circovirus type 2 LN strain is characterized in that the microorganism preservation number is CGMCC No.47094.
  2. 2. The haemophilus parasuis serum 4 type HB strain is characterized in that the microorganism preservation number is CGMCC No.46698.
  3. 3. The haemophilus parasuis serum 5 type JX02 strain is characterized in that the microorganism preservation number is CGMCC No.46699.
  4. 4. The method for preparing the suspension cell adapted strain of the porcine circovirus type 2 LN strain, which is characterized by comprising the steps of carrying out suspension culture on PK15 cells, replacing a maintenance solution after sedimentation of the cells when the concentration of the PK15 suspension cells reaches more than 2X 10 6 cells/ml, and inoculating 6% -10% of the porcine circovirus type 2 LN strain, and carrying out suspension culture for 96-120 hours to obtain a virus solution.
  5. 5. The method according to claim 4, wherein the PK15 cells are subjected to suspension culture under the conditions that the cell inoculation density is 0.5X10 6 ～1.0×10 6 cells/ml, the culture parameters are 36-38 ℃, the stirring speed is 100r/min, the pH value is 7.2-7.4, the dissolved oxygen value is 50%, the antifoaming agent is added by 0.1%, the culture medium is a Virus Pro serum-free culture medium or a VP SFM AGT serum-free culture medium, and the parameters of suspension culture after inoculating porcine circovirus type 2 LN strain are the culture temperature is 36-38 ℃, the pH value is 7.2-7.4, the dissolved oxygen value is 50%, the antifoaming agent is added by 0.1%, and the rotation speed is 100r/min.
  6. 6. An inactivated vaccine of porcine circovirus type 2, haemophilus parasuis serotype 4 and haemophilus parasuis serotype 5, comprising a prophylactically or therapeutically effective amount of an inactivated antigen of porcine circovirus type 2 LN strain according to claim 1, an inactivated antigen of haemophilus parasuis serotype 4 HB strain according to claim 2 and an inactivated antigen of haemophilus parasuis serotype 5 JX02 strain according to claim 3, together with an immunologically acceptable adjuvant.
  7. 7. The inactivated vaccine as claimed in claim 6, wherein the volume ratio of the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum type 4 HB strain and the inactivated antigen of the haemophilus parasuis serum type 5 JX02 strain is (1-1.5): (1-2), preferably the volume ratio of the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum type 4 HB strain and the inactivated antigen of the haemophilus parasuis serum type 5 JX02 strain is (1-2): 1:1, most preferably the volume ratio of the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum type 4 HB strain and the inactivated antigen of the haemophilus parasuis serum type 5 JX02 strain is 1:1:1.
  8. 8. The method for preparing the inactivated vaccine as claimed in claim 7, comprising: The preparation method comprises the steps of (1) respectively preparing an inactivated antigen of a porcine circovirus type 2 LN strain, an inactivated antigen of a haemophilus parasuis serum type 4 HB strain and an inactivated antigen of a haemophilus parasuis serum type 5 JX02 strain, (2) mixing the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum type 4 HB strain and the inactivated antigen of the haemophilus parasuis serum type 5 JX02 strain to obtain a water phase, and (3) mixing and emulsifying the water phase and an oil phase adjuvant to obtain the recombinant strain.
  9. 9. The method according to claim 8, wherein the water phase is obtained by mixing the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum 4 type HB strain, the inactivated antigen of the haemophilus parasuis serum 5 type JX02 strain, and the lentinan in a volume ratio of 1:1:1 (0.01-0.05), and preferably the water phase is obtained by mixing the inactivated antigen of the porcine circovirus type 2 LN strain, the inactivated antigen of the haemophilus parasuis serum 4 type HB strain, and the inactivated antigen of the haemophilus parasuis serum 5 type JX02 strain, and the lentinan in a volume ratio of 1:1:1 (0.03-0.05).
  10. 10. The method of claim 8, wherein the aqueous phase and the oil phase adjuvant are mixed according to a volume ratio of 3:1, and wherein the oil phase adjuvant is Montanide TM ISA 15AVG adjuvant.

Description

Porcine circovirus 2-type haemophilus parasuis disease bivalent inactivated vaccine and preparation method thereof Technical Field The invention relates to a bivalent vaccine for animals, in particular to a bivalent inactivated vaccine for porcine circovirus type 2 and haemophilus parasuis disease and a preparation method thereof, belonging to the field of bivalent inactivated vaccines for porcine circovirus type 2 and haemophilus parasuis disease and preparation thereof. Background Porcine circovirus (Porcine circovirus, PCV), belonging to the genus circovirus of the family circoviridae, is a non-envelope, single-stranded DNA virus. It has two genotypes of PCV1 (PCV 1) and PCV2 (PCV 2), wherein PCV1 is non-pathogenic, but widely exists in pig bodies and pig source cell lines, PCV2 is pathogenic, and can cause various diseases of pigs, such as postweaned pig multisystemic failure syndrome (PMWS), pig Dermatitis and Nephrotic Syndrome (PDNS), congenital Tremor (CT) of newborn pigs, pig reproductive disorder, granulomatous enteritis, pig respiratory syndrome and the like. PCV2 infection is mainly characterized by progressive emaciation, slow growth, dyspnea, skin blushing, yellow dyeing, anorexia, mental depression, mao Pengluan, morbidity of 4-25% and mortality rate of about 25-40% of piglets at 8-12 weeks after weaning. At present, PMWS occurs in pig farms all over the world, so that huge economic loss is caused for pig industry, and development of pig industry is severely restricted. PCV2 antibodies were detected in pig groups in Beijing, hebei, shandong, tianjin, jiangxi, jilin, henan and other places in China, and the positive rate was as high as 51%. Serological investigation of the PCV2 infection of the large-scale pig farm by the scholars shows that at least 29% of pig groups in part of pig groups are positive for antibodies, and the positive rate of part of pig groups is as high as 100% and the pig groups tend to rise year by year. Haemophilus parasuis (Haemophilus parasuis, HPS) is a common bacterium of the upper respiratory tract of pigs and is the causative agent of swine cellulose polyase serositis (Glasser's disease). With the development of large-scale and intensive pig farms, infectious diseases characterized by multiple serositis, arthritis, dyspnea, high fever and high mortality of pigs caused by HPS bring about great economic losses to the pig industry, and diseases caused by HPS have gradually become important diseases worldwide. The foreign epidemiological investigation of HPS shows that the separation rate of the clinical pigs is about 20 percent, and the serotypes with relatively strong toxicity, such as serotypes 5, 4 and 13, are popular. Domestic epidemiological studies have shown that the dominant serotypes are type 4 and type 5. Currently, HPS has become an important pathogen affecting the healthy development of the pig industry, and has received increasing attention. Numerous literature reports and clinical surveys indicate that mixed infection with PCV and HPS is extremely widespread, with severe morbidity, resulting in serious economic losses to the pig industry. The prevention and treatment of porcine circovirus type 2 and haemophilus parasuis are highly valued, and vaccination is an important method of prevention, except for conventional biosafety measures. At present, although the porcine circovirus type 2 and haemophilus parasuis bivalent inactivated vaccine is sold in the domestic and foreign markets, the used strains have a certain difference from the strains which are popular in the field, and the strains which are popular in the field cannot be completely protected, so that improvement is needed. Disclosure of Invention The invention aims at providing a preparation method of a locally popular porcine circovirus type 2 LN strain and a suspension cell adaptation strain thereof; the second purpose of the invention is to provide a serum type 4 strain of the haemophilus parasuis which is popular in the field; the third object of the invention is to provide a locally popular haemophilus parasuis serotype 5 strain; The fourth purpose of the invention is to provide porcine circovirus type 2, haemophilus parasuis serum type 4 and haemophilus parasuis serum type 5 inactivated vaccines and a preparation method thereof; the above object of the present invention is achieved by the following technical solutions: The invention provides a porcine circovirus type 2 LN strain which is popular in the field, the microorganism preservation number of the strain is CGMCC No.47094, the classification name of the strain is that the porcine circovirus type 2 LN strain is preserved for 2025 and 12 months, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Star Xiyu No. 1 national academy of sciences of China, the Korean region of Beijing, the microbiological culture Collection center. The invention provides a serum 4 type HB strai