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目的表达2型猪链球菌(Streptococcus suis 2)调控因子AdcR蛋白,为研究其在2型猪链球菌中调控组氨酸三聚体家族蛋白的作用机制奠定基础。方法通过与相关家族蛋白进行同源性比较,在2型猪链球菌05ZYH33全基因组序列中找出编码AdcR的基因。以AdcR基因序列为基础,利用Primer5.0设计引物,采用PCR扩增AdcR基因,经BamHⅠ和HindIII双酶切后将目的片段通过T4DNA连接酶连接至表达载体pET32a,转化大肠埃希菌E.coli BL21,加入IPTG至终浓度为0.1mmol/L,37℃诱导4h表达AdcR蛋白,然后利用His亲和层析柱纯化AdcR蛋白。结果通过Blast分析,从2型猪链球菌05ZYH33全基因组序列中找到编码AdcR的编码基因05SSU0109。以合成的特异引物PCR扩增出469bp的AdcR片段,连接原核表达载体pET32a后转化E.coli,经IPTG 37℃诱导4h,成功表达出AdcR蛋白,其分子质量单位为40ku,与理论值相符。经His-Tag亲和层析纯化,获得较高纯度的重组AdcR蛋白。结论在大肠埃希菌中成功表达了可溶性的AdcR重组蛋白,为AdcR在2型猪链球菌中调控组氨酸三聚体家族蛋白作用机制研究奠定了基础。
Objective To express AdcR protein of Streptococcus suis 2 (type 2) and lay a foundation for the study of its mechanism of action in the regulation of histidine trimer family protein in Streptococcus suis type 2. Methods The gene encoding AdcR was found in the genome sequence of Streptococcus suis type 2 Streptococcus suis 05ZYH33 by homology comparison with related family proteins. Based on the AdcR gene sequence, Primer5.0 was used to design the primers. The AdcR gene was amplified by PCR. After digested with BamHⅠ and HindIII, the target fragment was ligated into the expression vector pET32a by T4 DNA ligase and transformed into Escherichia coli E.coli BL21, IPTG was added to a final concentration of 0.1 mmol / L, AdcR protein was induced for 4h at 37 ℃, then AdcR protein was purified by His affinity chromatography. Results The gene coding for AdcR, 05SSU0109, was found from the whole genome sequence of Streptococcus suis type 2 Streptococcus suis type 2 by Blast analysis. The 469bp fragment of AdcR was amplified by PCR. The recombinant plasmid pET32a was inserted into prokaryotic expression vector pET32a and transformed into E.coli. After induced by IPTG at 37 ℃ for 4h, AdcR protein was successfully expressed. Its molecular mass unit was 40ku, which was consistent with the theoretical value. Purification by His-Tag affinity chromatography to obtain higher purity recombinant AdcR protein. Conclusions The soluble recombinant AdcR protein was successfully expressed in Escherichia coli, which lays the foundation for the study of the mechanism of AdcR regulating the protein of histidine trimer family in Streptococcus suis type 2.