禽流感病毒M2e基因与鸡IgG Fc基因在巴斯德毕赤酵母中的融合表达  

Expression of a fusion protein of influenza virus matrix 2 protein ectodomain segments(M2e) and avain IgG Fc in Pichia pastoris

作　　者：尚书文[1] 侯继波[2] 徐公豹[2] 牛明福[1] 王秀清[1] 何家惠[2] 陈溥言[1] 姜平[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点实验室,江苏南京210095 [2]江苏省农业科学院兽医研究所,江苏南京210014

出　　处：《西北农林科技大学学报（自然科学版）》2008年第1期17-22,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：江苏省科技厅高技术项目(BG2006324)

摘　　要：【目的】得到融合蛋白M2e-Fc,并检测其免疫学特性,为研制禽流感通用疫苗奠定基础。【方法】根据禽流感病毒H5N1的M2e蛋白氨基酸序列设计2条长互补引物,通过重叠区互补扩增基因法扩增出目的基因M2e,然后与鸡IgGFc片段基因一起克隆入毕赤酵母表达质粒中,构建酵母表达载体。将阳性质粒线性化后电转化入感受态毕赤酵母X-33中,再经甲醇诱导后,通过Tricine-SDS-PAGE电泳及Western-blotting鉴定融合蛋白。之后用其免疫非免疫鸡,检验其免疫原性。【结果】成功构建了表达M2e和Fc融合蛋白的酵母表达载体pPICZαA-M2-Fc,并通过Zeocin筛选及PCR鉴定出了阳性重组子;阳性重组子经甲醇诱导后得到融合蛋白,Tricine-SDS-PAGE电泳及Western-blotting鉴定证实,融合蛋白得到正确表达,并且可以与M2e阳性血清反应。动物免疫试验证实,该融合蛋白可以诱导鸡产生抗M2e抗体,具有良好的免疫原性。【结论】融合蛋白M2e-Fc在巴斯德毕赤酵母表达系统中得到了成功表达,该融合蛋白具有较好的免疫原性,为后期进行其他免疫试验奠定了基础。[Objective] The study is to abtain a fusion protein M2e-Fc and to learn its immunogenicity. [Method] According to the partiality codon of Pichia pastoris,influenza virus matrix 2 protein ectodomain segments （M2e） was amplified by SOE PCR using a couple of long primers. Then M2e gene and avain IgG Fc segments gene were digested and inserted into Pichia pastoris expression vector pPICZaA, and the ex- pression plasmid pPICZαA-M2-Fc was obtained. The recombinant plasmid was lineared and transformed in- to competant Pichia pastoris X-33 strain by electroporation. Then the recombinant transformants were selected by Zeocin and identified by PCR using the 3＇AOX and 5＇AOX primers. The expression of the fusion protein in yeast cells was induced by addition of methanol every 24 hours to 1% , and was analyzed by Tricine-SDS-PAGE and Western blotting. [Result] The fusion expression vector for M2e and Fc segments was successfully constructed, and the relative molecular mass of the fusion protein was measured to be 23kDa. The fusion protein was expressed at a yield of 321 mg per liter of culture with a specific antigenicity as demonstrated by Western blotting. After immunized chicken with M2e-Fc,anti-M2e antibody titer was determined by indirect ELISA. [ConclusionI The fusion protein M2e-Fc was successfully expressed and had good antigenicity.

关 键 词：禽流感病毒 M2e基因 鸡IGG Fc基因 巴斯德毕赤酵母 

分 类 号：S852.659.5[农业科学—基础兽医学] Q786[农业科学—兽医学]