牛呼吸道合胞体病毒G蛋白抗原区原核表达及反应原性鉴定

doi:10.16431/j.cnki.1671-7236.2021.09.036

摘要/Abstract

摘要： 本研究旨在获得重组牛呼吸道合胞体病毒（BRSV） G蛋白并对其进行反应原性鉴定。从NCBI上找出G基因的核苷酸序列，分析其抗原区域，利用Primer Premier 5.0软件设计1对特异性引物，利用RT-PCR方法扩增BRSV G基因片段，将目的片段连接到克隆载体pMD19-T后，对其进行双酶切鉴定和测序。将双酶切后的目的片段进行胶回收，构建重组质粒pET-32a-G，将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，用Ni-IDA亲和层析法纯化经IPTG诱导表达的蛋白，并测定其浓度；通过SDS-PAGE和Western blotting方法对该蛋白进行分析与鉴定。结果显示，本试验成功克隆出大小约为567 bp的G基因，获得分子质量约为40 ku的可溶性重组蛋白，优化诱导条件后用SDS-PAGE对表达产物进行鉴定，在16℃、IPTG浓度1.2 mmol/L、诱导4 h时蛋白表达量最大；通过Western blotting检测发现，重组蛋白可与山羊源BRSV标准阳性血清发生特异性反应，说明该蛋白具有反应原性。综上，本研究成功表达并纯化了BRSV G蛋白，为建立BRSV抗体间接ELISA检测方法和BRSV亚单位疫苗的研制奠定了基础。

关键词: 牛呼吸道合胞体病毒(BRSV); G蛋白; 原核表达; 可溶性表达; 反应原性

Abstract: The purpose of this study was to obtain recombinant G protein of Bovine respiratory syncytial virus (BRSV) and identify its reactogenicity. The nucleotide sequence of G gene was found from NCBI, its antigenic region was analyzed, and a pair of specific primers was designed with Primer Premier 5.0 software. The BRSV G gene fragment was amplified by RT-PCR and ligated to the cloning vector pMD19-T, and then it was identified by double enzyme digestion and sequenced. The gel was recovered from the target fragment digested by double enzyme, then the recombinant plasmid pET-32a-G was constructed and transformed into E. coli BL21(DE3) competent cells. The protein induced by IPTG was purified by Ni-IDA affinity chromatography and its concentration was determined, then the protein was analyzed and identified by SDS-PAGE and Western blotting. The results showed that the G gene with the size of 567 bp was successfully cloned and the soluble recombinant protein with molecular weight of 40 ku was obtained. After optimizing the induction conditions, the expressed product was identified by SDS-PAGE. The protein expression was the highest at 16 ℃, IPTG concentration 1.2 mmol/L and induction for 4 h. Through Western blotting detection, it found that the recombinant protein could react with goat BRSV standard positive serum specifically, indicating that the protein had reactivity. In summary, BRSV G protein was successfully expressed and purified in this study, which laid a foundation for the establishment of indirect ELISA detection of BRSV antibody and the development of BRSV subunit vaccine.

Key words: Bovine respiratory syncytial virus (BRSV); G protein; prokaryotic expression; soluble expression; reactogenicity

中图分类号:

S852.65⁺3

武春霞, 周雅坪, 郭婷, 崔琦, 王宇琛, 田广原, 思汗, 孙衍立, 郝永清. 牛呼吸道合胞体病毒G蛋白抗原区原核表达及反应原性鉴定[J]. 中国畜牧兽医, 2021, 48(9): 3438-3446.

WU Chunxia, ZHOU Yaping, GUO Ting, CUI Qi, WANG Yuchen, TIAN Guangyuan, Sihan, SUN Yanli, HAO Yongqing. Prokaryotic Expression and Reactogenicity Identification of G Protein Antigen Region of Bovine Respiratory Syncytial Virus[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3438-3446.

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