人3型副流感病毒HN基因的克隆、表达及其免疫原性  

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

作　　者：白慕群[1] 安静[1] 安红[1] 包红[1] 周旭[1] 

机构地区：[1]兰州生物制品研究所第二研究室,兰州730046

出　　处：《中国生物制品学杂志》2008年第12期1054-1057,共4页Chinese Journal of Biologicals

摘　　要：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。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 nucleotide and amino acid sequences of amplified HPIV-3 HN gene to those of reference gene were 95% and 97% respectively. Compared 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 immunogenicity and induced high antibody response in mice.

关 键 词：人3型副流感病毒 HN基因 克隆 原核表达 免疫原性 

分 类 号：R373.13[医药卫生—病原生物学] Q78[医药卫生—基础医学]