猪细小病毒NS基因原核表达与多克隆抗体制备

旨在制备抗猪细小病毒(PPV)非结构蛋白共有氨基酸的NS多肽多克隆抗体。根据GenBank(MK993540)公布的PPV基因组序列,克隆其非结构蛋白NS1、NS2共有基因序列(NS基因),并进行生物信息学分析。进而将NS基因克隆至原核表达载体pET-32a,构建重组质粒pET-32a-NS,转化至大肠埃希菌Rosetta(DE3)中进行诱导表达,利用镍柱亲和层析技术纯化表达的重组多肽,用重组多肽免疫Balb/c小鼠制备多克隆抗体。结果显示,PPV杨凌株NS基因长258bp,编码86个氨基酸的多肽,是具有亲水性的非跨膜NS多肽,具有大量的B细胞线性表位。NS多肽在37℃、0.8mol/L IPTG条件下,诱导6h有大量的可溶性表达。免疫印迹试验结果显示,该多肽具有较好的抗原性。用纯化NS多肽免疫小鼠后获得鼠抗NS多肽的血清抗体效价为1∶12800。用制备的NS多克隆抗体检测纯化的NS重组多肽及真核表达的NS1蛋白,均能检测出相应特异性条带,为进一步研究NS蛋白在PPV致病过程中的作用奠定了基础。

Prokaryotic Expression of Porcine Parvovirus NS Gene and Preparation of Polyclonal Antibody

The objective of this study was to prepare polyclonal antibodies against non-structural protein NS of porcine parvovirus(PPV).According to the PPV genome sequence published in GenBank(MK993540),the NS gene of the nonstructural protein NS1,NS2 consensus sequence was cloned and analyzed by bioinformatics.The NS gene was subcloned into the prokaryotic expression vector pET-32 ato construct the recombinant plasmid pET-32 a-NS,and transformed into E.coli Rosetta(DE3)for expression.The fusion polypeptide purified by nickel column affinity chromatography were used to immunize Balb/c mice to prepare polyclonal antibodies.The results showed that the NS gene of PPV Yangling strain was 258 bp and encoded a polypeptide of 86 amino acids,which was a hydrophilic non-transmembrane polypeptide with a large number of B-cell linear epitopes.A lot of soluble polypeptides were expressed under the induction of 0.8 mol/L IPTG and 6 hat 37℃.The results of Western blot showed that the polypeptide had good immunogenicity.The serum antibody titer of the mouse anti-NS polypeptide obtained by immunizing mice with purified NS polypeptide reached 1∶12800.The purified NS fusion polypeptide and the eukaryotic expression proteins NS1 were detected by the prepared NS polyclonal antibody,and the specific corresponding bands were generated.The study laid the foundation for further study of the function of NS peptide in its pathogenesis.