猪圆环病毒2型灭活疫苗生产质量影响因素研究 摘 要 猪圆环病毒2型（PCV2）感染是造成断奶仔猪多系统衰竭综合征（PMWS）的主要原因,其它协同因素可增强疾病的严重程度。1991年加拿大首次爆发本病，随后世界上许多国家和地区都有本病的报道。本病以断奶仔猪和育肥猪生长缓慢、呼吸急迫、消瘦、贫血和出现黄疸现象等为特征，给养猪业造成严重经济损失。经血清学调查和病毒分离鉴定证实，本病在我国也广泛存在。 疫苗是预防此病的主要措施之一。为了有效地预防PCV2感染，本研究利用南京农业大学分离鉴定的PCV2-SH株作为疫苗毒株，进行了灭活苗的研究，为我国预防和控制该病奠定了基础。具体研究内容如下： 1．猪圆环病毒2型规模化增殖影响因素研究 PCV2体外培养主要采用PK15 细胞，本研究对细胞冻存复苏条件、血清、细胞消化传代方法、接毒和收获方法等进行了条件优化，结果为：复苏PK15细胞采用39℃的温水60秒内迅速溶解，细胞存活率最好；冻存细胞加入10%~15%二甲基亚砜，并采取4℃30 min →- 20℃2h→-70℃12h→液氮的冻存方法较理想；不同批次的小牛血清对细胞生长影响较大，选择含8%~10%新生血清的DMEM作为细胞生长液，均能保证细胞正常生长；胰酶消化前采用hank’s液洗涤细胞，使用1：4胰酶-EDTA消化液消化，按1:3-1:4的比例传代，细胞生长状态良好。采用同步接毒方式，收获的抗原病毒含量最高；每个15L转瓶的种毒接毒量为106.5TCID50最为适宜；病毒细胞维持液采用3%-5%血清的MEM培养基，含毒细胞的生长良好；含毒细胞收获时经-20℃冻融1-3次，病毒含量较高，从而为PCV2灭活疫苗制备提供了依据。 2．猪圆环病毒2型灭活与乳化工艺影响因素研究 取PCV2培养物用不同浓度甲醛37℃作用24小时，用0.2%浓度甲醛作用不同时间，分别取样用PCR和间接免疫荧光法检测灭活效果，结果为：用0.2%浓度甲醛37℃灭活20小时可完全灭活圆环病毒。用法国进口206佐剂为油相，PCV2 SH株灭活抗原为水相,将油相、水相按54:46的体积比混合到一起进行乳化，通过对乳化过程中的温度、PH值、搅拌速度、搅拌时间、降温方式等进行试验，然后对成品苗进行乳化效果的检测和判定，结果为：当水相的PH值为8.2，水相、油相温度为32℃，搅拌速度为50HZ，搅拌时间为30分钟时乳化效果最好。将按上述方法乳化合格的3批PCV2灭活疫苗按质量标准进行物理性状和无菌检测，均符合要求。 3．猪圆环病毒2型灭活苗安全性与免疫效力试验 选择14～21日龄仔猪，用3批灭活疫苗接种，每头肌肉注射疫苗4ml，进行安全性检验，连续观察14日，均无异常临床反应。免疫两次，每次免疫1ml，两周后加强免疫一次，于首次免疫后1、2、3、4周采血,分离血清，用间接ELISA测定PCV2抗体，结果为：3批灭活疫苗免疫猪体后都可以检测到特异性抗体，免疫2周后开始产生抗体，免疫4周抗体水平达到最高，而非免疫组始终未检测到特异性抗体。采用5～6周龄清洁级小白鼠免疫，每只0.2ml，两周后按相同途径和剂量进行第2次接种，首免后5周采血，分离血清，测定血清中PCV2 ELISA抗体，小鼠免疫后抗体平均效价不低于1∶800。 关键词：猪圆环病毒2型；灭活疫苗；生产质量；影响因素

STUDY ON THE INFLUENCING FACTORS OF PRODUCTION QUALITY OF INACTIVATED VACCINE OF PORCINE CIRCOVIRUS TYPE 2 ABSTRACT Porcine circovirus 2（PCV2）is the causal agent of postweaning multisystemic wasting syndrome（PMWS）. The syndrome mainly affects weaned pigs and is characterized by dyspnea, progressive weight loss, respiratory signs and jaundice.The disease was first described in Canada in 1991 and now is considered to be widespread throughout the most importmant swine production areas of the world. The serological investigations and virus isolation and identification confirmed that the disease is also widespread in China. The ministration of PCV2 vaccine is one of the main measures to prevent this disease. In this study the PCV2-SH strain which was isolation and identificated by Nanjing Agricultural University, was used as the vaccine strain for studying on the factors affecting the inactivated vaccine production quality. It should be helpful to prevent and control this disease in our country. The main contents are as follows: 1．Study on the factors influencing large-scale proliferation of porcine circovirus type 2 Porcine circovirus 2（PCV2）is mainly cultured in PK15 cells. Parallel experiments based on different conditions of recovering and freezing, serum concentration, digesting and passaging method, inoculation and harvesting method had been done. The results showed that: the cells could grow well when they were thawed at 39 ℃ water bath within one minute; Use of 10%-15% DMSO and the procedure of 4℃30min→-20℃ 2h→-70℃ 12h→Liquid nitrogen was the best freezing program; Different batches of fetal bovine serum could influnce the growth of PK15 cells greatly; DMEM supplemented with 8%-10% fetal serum（FBS）as cell growth medium could ensure the normal growth of cells; the best cell dispersion and growth result could be achieved by washing away the serum residue with Hanks solution, digesting with trypsin-EDTA at the ratio of 1:4 and passaging at the ratio of 1:3-1:4. Viral stock with higher titers could be obtained with synchronal inoculation method; optimum inoculation dosages of PCV2 was 106.5TCID50 for each 15L-roller-bottle; MEM with 3%-5% fetal bovine serum（FBS）was the optimum medium for virus maintenance, which could promote the growth of infected cells; cell culture supernants with higher viral titer could be obtained by freezing and thrawing 1-2 times. 2．Study on the influencing factors of inactivated and emulsification process of porcine circovirus type 2 Virus solution was inactivated with different concentrations of formaldehyde at 37 ℃ for 24 hours and also with 0.2% formaldehyde at 37 ℃ for different times, the samples were collected to detect inactivation effect by PCR and indirect immunofluorescence assay. The results showed that PCV2 could be completely inactivated with 0.2% formaldehyde at 37℃ for 20 hours. ISA 206 was used as oil phase and inactivated PCV2 was used as water phase which were mixed at the ration of 54:46 in volume. Parallel experiments had been done based on different temperature, PH, mixing speed, mixing time and temperature deducing in the emulsification procedure. The effect of emulsification of the finished product had been tested and determined. The results showed that good emulsification effect was obtained when the PH of the water phase was 8.2, the temperature of both oil and water phase was 32℃, the mixting speed was 50HZ and the mixting time was 30 min. Virus solution was inactivated with the methods mentioned above and used to make three batches of vaccines. Physical characters and sterility of the vaccine were detected and results showed that all of them met the requirements. 3．Safety and protective efficacy experiments of porcine circovirus type 2 To test the safety of vaccines, 14 to 21 days old piglets were injected intramuscularly with three batches of inactivated vaccine, 4ml each piglet. No abnormal clinical reaction occurred during 14 days after injection. Then piglets were injected with 1ml different batches of inactivated vaccine and boosted two weeks later with the same method. Serum was separated at 1, 2, 3 and 4 weeks after the first immunization to detect PCV2 speific antibody by indirect ELISA. The results showed that antibodies could be detected after injection with all the three batches of inactivated vaccine. The antibodies could be detected two weeks after the first injecion and peaked at four weeks. No specific antibody was detected in control group. In addition, 5-6-week-old clean grade mice were injected with 0.2ml inactivated vaccine and reinjected by the same method two weeks later. Serum was isoltated five weeks after the first immunization to detect the PCV2 antibody and the results showed that the average antibody titers were above 1:800. KEY WORDS：Porcine circovirus type2 ; Inactivated vaccine; Production quality; Influencing factors