猪流行性腹泻病毒截短M基因克隆、生物信息学分析及其截短片段表达

【目的】 探索猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)截短M蛋白的序列结构特征与原核表达情况。【方法】 根据已公布的PEDV M基因序列设计1对特异性引物,以从阳性病料提取的RNA为模板,通过RT-PCR扩增并克隆PEDV截短的M基因(rM),应用在线生物信息学软件预测rM蛋白的结构特征。将得到的截短的M基因克隆至原核表达载体pET-28a(+)中,构建原核表达质粒pET-28a-rM,将鉴定正确的pET-28a-rM转化大肠杆菌BL21(DE3)感受态细胞,成功构建重组菌株BL21(pET-28a-rM),并对重组菌株进行IPTG诱导表达,优化重组蛋白的表达条件,重组蛋白经亲和层析纯化后进行SDS-PAGE及Western blotting检测,同时应用重组蛋白制备兔抗rM多克隆抗体。【结果】 试验克隆得到大小为366 bp的截短的M基因,重组蛋白由121个氨基酸组成,预测蛋白分子质量大小约为12.8 ku。该蛋白的二级结构由无规则卷曲、延伸链、β-转角和α-螺旋组成,占比分别为48.76%、33.88%、9.09%和8.26%。该蛋白不含信号肽,但有跨膜区,包含21个磷酸化位点。SDS-PAGE结果显示,重组蛋白大小约为15 ku,以包涵体蛋白形式存在,在37 ℃、1 mmol/L IPTG诱导12 h时蛋白的表达量最高。Western blotting检测结果表明,重组蛋白与PEDV阳性血清具有较好的反应原性,纯化的重组蛋白免疫新西兰大白兔获得的高免血清效价高于1∶51 200。【结论】 本研究成功克隆PEDV 截短的M基因,对rM蛋白进行了生物信息学分析,获得了高纯度的rM蛋白,为猪流行性腹泻治疗及检测用生物制品的开发奠定了基础。

Cloning,Bioinformatics Analysis and Truncated Fragment Expression for Truncated M Gene of Porcine Epidemic Diarrhea Virus

【Objective】 The aim of this study was to investigate the sequence structure and prokaryotic expression of Porcine epidemic diarrhea virus (PEDV) truncated M protein.【Method】 A pair of specific primers were designed according to the published M gene sequence of PEDV.The truncated M gene (rM) of PEDV was amplified by RT-PCR and cloned using RNA extracted from positive clinical samples.The structural characteristics of rM protein were predicted by online bioinformatics software.The obtained truncated M gene was cloned into prokaryotic expression vector pET-28a(+),and the prokaryotic expression plasmid pET-28a-rM was constructed.The correctly identified pET-28a-rM was transformed into E.coli BL21(DE3) competent cells,and the recombinant strain BL21(pET-28a-rM) was successfully constructed,then the recombinant strain was induced by IPTG,the expression conditions of the recombinant protein were optimized,and the recombinant protein was purified by affinity chromatography and detected by SDS-PAGE and Western blotting.Meanwhile,rabbit anti-rM polyclonal antibody was prepared by using the recombinant protein.【Result】 The truncated M gene with a size of 366 bp was cloned.The recombinant protein was composed of 121 amino acids,and the predicted molecular weight was about 12.8 ku.The secondary structure of the protein was composed of random coil,extended strand,beta turn and alpha helix,and the components were 48.76%,33.88%,9.09% and 8.26%,respectively.The protein contained no signal peptide,but had transmembrane region,containing 21 phosphorylation sites.SDS-PAGE results showed that the size of recombinant protein was about 15 ku and existed with the form of inclusion body protein.The protein expression was the highest when induced at 37 ℃ with 1 mmol/L IPTG for 12 h.Western blotting result showed that the recombinant protein had good reactogenicity with the PEDV positive serum.The high immune serum with the titer higher than 1∶51 200 was obtained after the New Zealand White rabbits immunized with purified recombinant protein.【Conclusion】 This study successfully cloned the PEDV truncated M gene the bioinformatics analysis was carried out,and obtained high-purity rM protein,and established a material foundation for the development of biological products for porcine epidemic diarrhea treatment and detection.