猪细小病毒VP2基因在家蚕中的高效表达及免疫原性评估  

High Expression and Immunogenicity Evaluation of Porcine parvovirus VP2 Gene in Silkworm(Bombyx mori)

作　　者：占鹏飞 窦金萍 易咏竹 刘兴健[2] 李轶女[2] 冯世民 张志芳[2] 张金卫 ZHAN Peng-Fei;DOU Jin-Ping;YI Yong-Zhu;LIU Xing-Jian;LI Yi-Nyu;FENG Shi-Min;ZHANG Zhi-Fang;ZHANG Jin-Wei(Huzhou Agricultural Science and Technology Development Center,Huzhou 313000,China;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China;School of Biotechnology,Jiangsu University of Science and Technology,Zhenjiang 212100,China)

机构地区：[1]湖州市农业科技发展中心,湖州313000 [2]中国农业科学院生物技术研究所,北京100081 [3]江苏科技大学生物技术学院,镇江212100

出　　处：《农业生物技术学报》2023年第6期1275-1283,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32002236,32072796);宁夏回族自治区重点研发项目(2020BFG02017);湖州市科技计划项目(2019GZ18);市级农科院联盟区域示范性项目(2022SJLM10)。

摘　　要：猪细小病毒(Porcine parvovirus,PPV)可引起猪(Sus scrofa domesticus)繁殖障碍,疫苗是预防猪细小病毒病、提高母猪抗病力和繁殖率的有效方法。为高效表达猪细小病毒结构蛋白VP2基因及制备猪细小病毒基因工程亚单位疫苗,根据家蚕密码子频率将PPV-VP2的基因序列进行优化合成,并将此目的基因片段连接到转移载体,通过共转染将目的基因重组到orf1629基因缺损的亲本病毒BmBacmid上,获得重组杆状病毒(Recombinant baculovirus)rBmNPV(PPV-VP2)。将重组杆状病毒rBmNPV(PPV-VP2)感染家蚕(Bombyx mori)蚕蛹,并用表达的抗原制作疫苗,采用豚鼠(Cavia porcellus)进行疫苗效力试验。经Western blot和质谱分析、电镜观察表达产物。结果表明,在蚕蛹中成功表达出可自组装成病毒空衣壳颗粒的目的蛋白;表达蛋白产物经血凝反应测定,每克蚕蛹表达的抗原效价相当于200剂以上商用疫苗;用表达产物制备成3种抗原剂量的疫苗免疫豚鼠,均产生抗体反应,其抗体能在体外中和PPV病毒。即使每克蚕蛹制备200头份疫苗免疫豚鼠,其血凝抑制效价(hemagglutination inhibition,HI)也达到了1∶128,中和抗体效价达到1∶83,不低于市售商品苗免疫产生的效价。本研究为猪细小病毒亚单位空衣壳疫苗提供了低成本生产方法。Porcine parvovirus(PPV)can cause porcine(Sus scrofa domesticus)reproductive disorder.Vaccination is an effective method to prevent pigs from PPV disease and improve the immunity and reproductive rate of sows.In order to efficiently express PPV structural protein VP2 gene and prepare PPV genetic engineering subunit vaccine,the PPV-VP2 gene was optimized and synthesized according to silkworm(Bombyx mori)codon frequency in this study.The VP2 gene was cloned to transfer vector and recombined into the parental virus BmBacmid with orf1629 gene deficient by co-transfection.The recombinant baculovirus rBmNPV(PPV-VP2)was obtained and used to infect silkworm.The expressed antigen was used to prepare subunit vaccine,and guinea pigs(Cavia porcellus)were used for vaccine efficacy evaluation.The expressed recombinant protein was confirmed by Western blot and mass spectrometry.The observation of electron microscope indicated that the virus like particles(VLP)could be self-assembled into a similar size and morphology to PPV.The results of hemagglutination assay(HA)indicated that expressed antigen titer per gram of silkworm pupae was equivalent to more than 200 doses of commercial vaccine.Guinea pigs were immunized with 3 different doses of PPV-VP2 antigen contents,all inoculated animals had a seroconversion,and the antibody could neutralize PPV in vitro.If a guinea pig was immunized with 5 mg of silkworm pupae(parallel to 200 doses of antigen in commercial vaccine),the hemagglutination inhibition(HI)and neutralizing antibody titer of the guinea pig reached 1∶128 and 1∶83,respectively,equal to those of animals vaccinated with the commercial vaccine.This study provides a low cost production method for PPV genetic engineering subunit vaccine.

关 键 词：猪细小病毒(PPV) 病毒空衣壳颗粒(VLP) 杆状病毒表达系统 免疫效力 家蚕 

分 类 号：S855.3[农业科学—临床兽医学]