A组链球菌M蛋白重组多肽原核表达载体构建及融合蛋白的表达

Construction of a divalent polypeptide vaccine expression vector for M protein of group A streptococcus and inducible expression of GST fusion protein

目的:设计针对A组β溶血性链球菌M1和M12蛋白的复合多肽;构建含M蛋白基因(emm基因)1型和12型特异性抗原决定簇基因及其保守区J14肽基因的GST标签的重组表达载体;并诱导和优化GST融合蛋白的表达。方法:在NCBI Genebank和Oligo 6引物设计软件中选择分别编码M蛋白emm基因1型和12型信号肽后35个氨基酸及其相同的保守区14肽基因序列(全长270 bp),通过重叠PCR(Overlap PCR)合成所需的核苷酸序列,经测序确定序列完全和设计的相匹配后,将该重组片段克隆到pGEX-4T-1表达载体中,阳性克隆经双酶切和测序鉴定正确后建立稳定表达该重组质粒的大肠杆菌BL21株系(pE/B);通过聚丙烯酰胺凝胶的考马斯亮蓝染色和Western blot来检测在不同时间(0、2、6、8、18和24小时)和温度(25℃、30℃和37℃)以及不同浓度(0.01、0.1、1 mmol/L)异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导GST融合蛋白(GST/emm)表达情况,并对诱导表达的GST/emm切胶进行质谱分析鉴定。结果:成功构建含有emm1和emm12抗原决定簇及M蛋白保守区J14肽基因的原核表达载体;并诱导其稳定表达,在1 mol/L IPTG诱导6～8小时达高峰,不同温度下均有过量表达;质谱检测诱导蛋白GST/emm条带结果正确。结论:成功构建A组β溶血性链球菌的emm1型和emm12型原核表达载体,并稳定诱导GST/emm表达,为下一步研究GST/emm的纯化、酶切以及免疫原性的鉴定和疫苗研制打下夯实的基础。

Objective：To design a recombinant polypeptide vaccine specific to group A [3-hemolytic streptococcus serotypes M1 and M12 protein; and clone the gene sequences of the designed vaccine and construct a recombinant vector（pGEX-4T-l-emml/12- J14, pE） containing fragments of emml gene and emml2 gene that is the type specific epitopes of M protein and J14 sequence variants in the conserved C-terminal region of M protein; so that to induce the expression of GST fusion protein（GST/emm）. Methods：A re- combinant polypeptide vaccine was designed by linking 35 amino acids after signal peptide separately from M1 and M12 protein and a common conservative sequence J14 which showed certain immunogenicity but no cross reaction with human tissue protein in the order of M1-12-J14, and analyzed for homology of amino acids to that of human tissue protein by blast to NCBI data bank. A group of above oli- gonucleotides was synthesized by overlap PCR, in which the restriction sites of BamH I and Xho I were introduced at 5＇ and 3＇ termi- nus respectively. The synthetic sequence was digested with BamH I and Xho I and cloned into cloning vector pGEX-4T-1 （ pG）, and the constructed recombinant plasmid was identified by sequencing. Furthermore to induce and optimize the expression of GST/emm with IPTG at different times（2,6,8,18 and 24 h） or different concentrations（0.01 mmol/L,0.1 mmol/L and 1.0 mmol/L）and under dif- ferent temperatures （25 % ,30℃ and 37℃ ） ; last to check and identify the gel sample of GST/emm with MALDI-TOF. Results： 1. The gene sequence of designed vaccine was successfully cloned, and a recombinant cloning vector（ pGEX-4T-1-emml/12-J14 ,pE）carrying designed gene Sequence was obtained ;2. Using this plasmid, we have achieved over expression of soluble recombinant polypeptide as a GST fusion protein using E. coli BL21 strain, under optimized environmental factors such as culture time （the maximum during 6-8 h induced） and inducer （ItrrG） concentration （the optimal concentration 1.0 mmol/L ）, but it seems that induced temperature had no significant effects on the the expression of GST/emm（ under different temperature, GST/emm always over expressed ） ;3. GST/emm gel sample with designed amino acids was identified with SDS-PAGE or Western blot. Conclusion：We have first constructed a recom- binant expression plasmid （pGEX-4T-l-emml/12-J14, pE）, in which we merged recombinant polypeptide with the glutathione S-transferees （GST） coding sequence downstream of the tac-inducible promoter and successfully induce and optimize GST fusion protein ex- pression.