人3型副流感病毒HN基因的克隆、表达及其免疫原性

目的克隆人3型副流感病毒(HPIV-3)HN基因,构建原核表达质粒,并检测表达蛋白的免疫原性。方法从呼吸道感染患儿呼吸道分泌物中提取HPIV-3RNA,用套式RT-PCR扩增HN基因,克隆至pGEM-T载体上,进行核苷酸序列分析,并用生物信息软件分析HN蛋白的二级结构,构建截短的HN基因原核表达载体pET-28a-HN,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达蛋白经SDS-PAGE和Western blot检测后,进行纯化和复性。并以此蛋白免疫昆明小鼠,检测小鼠血清中HN抗体效价。结果扩增出的HPIV-3的HN基因核苷酸序列与参考序列的同源性为95%,氨基酸序列同源性为97%,有16处发生了突变。截短的HN基因的原核表达载体经酶切鉴定及测序,证明构建正确。重组HN蛋白的表达量占菌体总蛋白的30%以上,且具有良好的反应原性。复性的重组HN蛋白免疫的小鼠可产生高效价的抗HN抗体。结论原核表达的截短的HN蛋白具有良好的免疫原性,能够刺激小鼠产生较高水平的抗体应答。

Cloning and Expression of HN Gene of Human Parainfluenza Virus Type 3 and Immunogenicity of Expressed Product

Objective To clone the HN gene of human parainfluenza virus type 3(HPIV-3),construct prokaryotic expression vector and study the immunogenicity of expressed HN protein.Methods HPIV-3 RNA was extracted from the secrete of children with lower respiratory tract infection for amplification of HN gene by RT-PCR.The amplified HN gene was cloned into plasmid pGEM-T for nucleotide sequencing.The secondary structure of HN protein was predicted by bioinformatic software,based on which a prokaryotic expression vector pET-28a-HN for truncated HN gene was constructed and transformed to E.coli BL21(DE3)for expres-sion under induction of IPTG.The expressed protein was identified by SDS-PAGE and Western blot then purified and renaturalized.KM mice were immunized with the expressed protein and determined for serum HN antibody titer.Results The homologies of nu-cleotide and amino acid sequences of amplified HPIV-3 HN gene to those of reference gene were 95%and 97%respectively.Com-pared with reference gene,the amplified HPIV-3 HN gene showed mutation of amino acids at 16 sites.Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-HN for truncated HN gene was constructed correctly.The expressed product contained more than 30%of total somatic protein and showed good reactogenicity.The HN protein after renaturalization induced high antibody titer against HN in mice.Conclusion The truncated HN protein expressed in prokaryotic cells showed good immunogenici-ty and induced high antibody response in mice.