非洲猪瘟病毒p30生物信息学分析及多克隆抗体的制备  

Bioinformatics analysis and preparation of polyclonal antibody against African Swine Fever Virus p30

作　　者：陈春龙 张丽荣 孔令保[1,2] CHEN Chunlong;ZHANG Lirong;KONG Lingbao(College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China;Nanchang Key Laboratory of Animal Virus and Genetic Engineering,Nanchang 330045,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]南昌市动物病毒与基因工程重点实验室,江西南昌330045

出　　处：《生物灾害科学》2024年第4期524-535,共12页Biological Disaster Science

基　　金：国家自然科学基金项目(31460667、32360908);江西省自然科学基金重点项目(20224ACB205004);南昌市动物病毒与基因工程重点实验室(2021-NCZDSYS-008)。

摘　　要：【目的】目前国内流行的变异非洲猪瘟病毒(African swine fever virus,ASFV)表现出毒力低,传染性强,隐蔽性强的特点,非洲猪瘟(African swine fever,ASF)临床表现不明显,早期诊断难度大,快速灵敏的早期诊断试剂盒对于非洲猪瘟防控至关重要,而p30的研究及其多克隆抗体的制备可以为早期诊断试剂盒的开发提供重要的理论依据。【方法】根据中国农业科学院哈尔滨兽医研究所分离的非洲猪瘟毒株Pig/Heilongjiang/HRB1/2020(GenBank:MW656282)提供的p30的氨基酸序列,利用生物信息学软件对该序列的理化性质,亲/疏水性,跨膜区,信号肽及二三级结构进行分析预测,由生物公司优化密码子并合成重组质粒pET-28a-p30。将p30克隆到pET-32a(+)原核表达载体中,将其转化至E.coli BL21(DE3)感受态细胞,对异丙基硫代半乳糖苷(IPTG)诱导浓度、诱导温度及诱导时间进行优化,用纯化所得的p30蛋白对小鼠进行3次免疫,获取多克隆抗体并检测效价。【结果】由生物信息学分析结果得出p30蛋白含194个氨基酸,分子式为C_(1001)H_(1575)N_(259)O_(308)S_(7),p30蛋白为亲水性蛋白,不含跨膜区,无信号肽,二三级结构中含大量的α-螺旋。蛋白表达结果显示,当诱导时间为6 h,IPTG终浓度为0.05 mmol/L,诱导温度为34℃时,p30重组蛋白表达量达到最佳,浓度高达0.9 mg/mL以上,1 L菌液可产53 mg重组蛋白。通过间接ELISA测得小鼠血清抗体效价高达1:10000000。Western Blotting结果显示,制备的p30蛋白具有较好的特异性。【结论】经过表达优化p30蛋白产量得到明显提高,并获得效价较高的多克隆抗体,且具有很好的特异性。对研发针对p30更加灵敏的诊断试剂盒提供了重要的理论依据。[Objective]The mutated African swine fever virus(ASFV)currently circulating in China shows the characteristics of low virulence,strong infectivity and strong concealment.The clinical manifestations of African Swine Fever(ASF)are not obvious,and the early diagnosis is difficult.Rapid and sensitive early diagnostic kits are very important for the prevention and control of African swine fever,and the study of p30 and the preparation of polyclonal antibody can provide an important theoretical basis for the development of early diagnostic kits.[Method]According to the separation of Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,African swine fever virus(ASFV)Pig/Heilongjiang HRB1/2020(GenBank:MW656282)provided p30 amino acid sequence.By using bioinformatics software,physical and chemical properties of the sequence,hydrophilic/hydrophobic,across the membrane area,the signal peptide,as well as the secondary and tertiary structures were analyzed and predicted,and the codon was optimized and the recombinant plasmid pET-28a-p30 was synthesized by the biological company.p30 was cloned into pET-32a(+)prokaryotic expression vector and transformed into E.coli BL21(DE3)receptor cells.Then,the induction concentration,induction temperature and induction time of isopropyl galactoside(IPTG)were optimized.The purified p30 protein was used to immunize mice for three times.Polyclonal antibody was thus obtained and titer was detected.[Result]The results of bioinformatics analysis showed that p30 protein contained 194 amino acids,the molecular formula was C1001H1575N259O308S7,p30 protein was hydrophilic protein and did not contain transmembrane region,no signal peptide was found,and there contained a lot ofα-helix in the secondary and tertiary structure.The protein expression results showed that when the induction time was 6 h,the final IPTG concentration was 0.05 mmol/L;when the induction temperature was 34℃,the recombinant protein expression level of p30 reached the best;when the concentration was hig

关 键 词：非洲猪瘟病毒(ASFV) p30蛋白 生物信息学分析 原核表达 多克隆抗体 

分 类 号：S852.651[农业科学—基础兽医学]