TGF-β潜态相关肽编码序列的原核表达载体构建及表达产物的纯化与鉴定

Construction of Prokaryotic Expression Vector of TGF-β Latent-related Peptide Coding Sequence and Purification and Identification of the Expressed Product

转化生长因子β(TGF-β)是促肝纤维的细胞因子,TGF-β潜态相关肽(LAP)和TGF-β的活化有关。本研究提取人血液中总RNA,逆转录转为c DNA,以此为模板PCR目的片段,构建原核表达载体pET28a-LAP-B、pET28a-LAP-F,并在不同诱导温度、IPTG浓度、诱导时机下进行诱导表达,筛选最佳诱导条件,通过镍琼脂糖亲和层析纯化重组蛋白,获得的蛋白通过SDS-PAGE及Western blot鉴定,并检测重组蛋白的浓度。结果表明,LAP全长及截短型原核表达载体构建成功,筛选最佳诱导条件为37℃培养4 h,OD_(600)值为0.8时降温至16℃,加入0.3 mmol/L(LAP-B)/0.45 mmol/L(LAP-F)IPTG诱导16 h,大量培养后通过镍柱纯化和透析得到重组蛋白,经SDS-PAGE及Western blot鉴定为LAP全长及截短型重组蛋白,并测得LAP-B、LAP-F重组蛋白浓度分别为637.6μg/mL、126.6μg/m L。本实验成功得到LAP全长及截短型重组蛋白,为研究LAP对TGF-β促纤维化作用的影响提供了实验基础。

Transforming growth factorβ(TGF-β)is a cytokine promoting liver fibrosis.TGF-βlatent associated peptide(LAP)is related to the activation of TGF-β.In this study,the total RNA in human blood was extracted and reverse transcribed into c DNA and used as a template for PCR amplification of the target fragments.The prokaryotic expression vector pET28 a-LAP-B,pET28 a-LAP-F was constructed and induced at different induction temperature,IPTG concentration and induction time.The best induction conditions were screened.The recombinant protein was purified by Ni-NTA agarose affinity chromatography.The obtained proteins were identified by SDS-PAGE and Western blot and the concentration of the recombinant protein was detected.The results showed that the prokaryotic expression vector of full-length and truncated LAP was successfully constructed.The optimal induction conditions were culture at 37℃for 4 h,OD_(600) value 0.8,and the expression was successfully induced by adding 0.3 mmol/L(LAP-B),0.45 mmol/L(LAP-B)IPTG at 16℃for 16 h.After a large amount of culture,the recombinant protein was obtained by purification and dialysis of the nickel column,and the total length of LAP and the truncated recombinant protein were identified by SDS-PAGE and Western blot.The concentrations of LAP-B,LAP-F recombinant protein were 637.6μg/mL and 126.6μg/m L respectively.LAP full-length and truncated recombinant protein were successfully obtained in this study,which provided an experimental basis for the study of the effect of LAP on the effect of TGF-β.