猪链球菌2型硫氧还蛋白还原酶多克隆抗体制备与亚细胞定位分析

Preparation of polyclonal serum and subcellular localization analysis of thioredoxin reductase in Streptococcus suis type 2

目的制备猪链球菌2型(Streptococcus suis,SS2)硫氧还蛋白还原酶(SsTrxR)抗体,分析其在SS2中的定位。方法对猪链球菌2型trxR基因进行生物信息学分析,PCR扩增TrxR基因,酶切、连接,构建重组表达载体pET30a-trxR。IPTG低温诱导表达SsTrxR重组蛋白,镍柱亲和纯化目的蛋白。皮下接种SsTrxR至日本大耳白兔,获取多克隆抗血清。Western blot分析SsTrxR抗血清特异性与在SS2中的定位。结果SsTrxR因子与细菌硫氧还蛋白还原酶的氨基酸序列存在多个保守区域与位点,同属内其它代表性链球菌硫氧还蛋白还原酶氨基酸序列同源性在68.5%以上,拥有Cys-Xaa-Xaa-Cys基序。成功构建pET30a-trxR表达质粒,SsTrxR原核蛋白呈可溶性表达,过镍柱纯化,获得目的蛋白。Western blot检测结果显示SsTrxR与抗血清反应性好,SsTrxR因子分布于细菌胞浆内。结论猪链球菌2型存在硫氧还蛋白还原酶SsTrxR,制备的兔源SsTrxR多克隆抗体特异性好,且该因子为胞内蛋白,为开展TrxR因子在SS2感染与免疫过程中发挥的功能研究奠定了基础。

Objective To express and prepare the recombinant thioredoxin reductase protein(SsTrxR)with its rabbit monoclonal antisera of Streptococcus suis type 2(S.suis type 2,SS2)and analyze its localization in SS2.Methods Bioinformatic analysis was conducted to align and predict the SsTrxR protein homology and subcellular localization.The trxR gene was amplified and cloned to the recombinant expression vector pET30 a.Then,the plasmid pET30 a-trxR was transformed into E.coli Rosetta(DE3)and prokaryotic expressed by IPTG.The recombinant protein was purified using Ni^(2+)affinity chromatography with 50 mmol/L imidazole cleaning buffer and 400 mmol/L imidazole elusion buffer,and subcutaneously immunized to obtain polyclonal antiserum in rabbits.Antiserum specificity and subcellular localization of SsTrxR were further determined by Western blotting.Results Multiple conserved amino acid sequences and a Cys-Xaa-Xaa-Cys motif of the SsTrxR were found as compared with the reported bacterial TrxR proteins.Sequence homology of the TrxR was above 68.5%between SS2 and other Streptococcus species.The recombinant expression plasmid of pET30 a-trxR was successfully constructed and the prokaryotic SsTrxR protein was purified.The molecular weight of the purified protein is 36 ku.The SsTrxR had good reactivity with the immuned rabbit antiserum.The SsTrxR factor was confirmed to distribute within the bacterial cytoplasm.Conclusion The thioredoxin reductase of SsTrxR is conservatively harbored and intracellular localized within SS2.The prepared rabbit anti-SsTrxR polyclonal antibody showed good reaction and specificity to this factor.All these data would contribute to further study on the biological functions evaluation of the SsTrxR factor in SS2 infection.