甲型流感病毒抗原表位融合多肽的原核表达及免疫原性分析  

Prokaryotic expression and immunogenicity analysis of multi-epitopefused peptide of influenza virus A

作　　者：马倩[1] 张玉娟[1] 史磊[1] 郑文明[1] 许君[1] 

机构地区：[1]河南农业大学生命科学学院,河南郑州450002

出　　处：《中国兽医学报》2014年第3期450-454,共5页Chinese Journal of Veterinary Science

基　　金：河南省科技攻关重点资助项目(102102110188)

摘　　要：根据甲型流感病毒多个株系的血凝素蛋白(hemagglutinin,HA)、基质蛋白(matrix,M1)和核蛋白(nucleoprotein,NP)的核苷酸序列的保守序列,筛选、设计并合成串联的DNA片段hmn,体外扩增hmn DNA,将之亚克隆至原核表达载体pET28α(+)中,转化宿主菌大肠杆菌BL21(DE3),0.3mmol/L的IPTG诱导后,HMN以包涵体形式表达。重组蛋白HMN复性后经镍柱亲和层析纯化,纯化后的蛋白可与特异性抗血清发生反应,纯化蛋白皮下注射免疫6~8周龄雌性小鼠,Western-blot检测抗体效价,显微观察小鼠脾脏和胸腺组织结构,流式细胞仪测定T淋巴细胞亚类数量,结果显示与对照组相比,HMN蛋白可明显刺激淋巴细胞增殖。本试验是对通用型流感多疫苗进行的有意义探索。Influenza viruses can cause an acute respiratory infectious disease to birds, humans and animals. Traditional vaccine has not played fast and effective protection function. A recombinant DNA sequence,hmn,was screened and designed according to the conservative polynucleotide se- quences of hemagglutinin （HA）,matrix （M1） and nucleoprotein （NP） of multiple influenza vi- rus. The hmn DNA fragment was amplified by PCR, sub-cloned into pET28a （＋） vector, and transformed host E. coli BL21 （DE3） cell. The recombinant protein HMN was mainly expressed as inclusion bodies after the induction of 0.3 mmol/L IPTG. Refolded HMN was purified by affin- ity chromatography. The purified HMN protein was subcutaneously injected into 6-8 week-old fe- male mice. The blood serum antibody titre was detected by Western-blot. After immunization,mi- croscopic observation was used to analyze the thymus and spleen organization structures. Flow cy- tometry detection showed the recombinant HMN protein could stimulate proliferation of splenic lymphocytes. This study is meaninl^ful exploration to l^et the universal influenza virus vaccine.

关 键 词：甲型流感病毒 抗原表位 通用疫苗 原核表达 动物免疫 

分 类 号：S851[农业科学—预防兽医学] Q812[农业科学—兽医学]