人跨膜蛋白39A原核表达载体构建、条件优化及可溶性表达

Construction of prokaryotic expression vector, optimization of expression conditions and soluble expression of human TMEM39A

目的构建人跨膜蛋白39A基因(TMEM39A)的原核表达载体,优化表达条件并获得可溶性表达的TMEM39A。方法以HEK293细胞的总RNA为模板,反转录PCR扩增TMEM39A,构建其原核表达载体pGEX-6P-1-TMEM39A,并在不同温度、IPTG浓度、A600nm值及时间条件进行诱导表达,然后利用上述获得的最佳诱导条件进行大量表达,分析其可溶性和抗原性。结果重组表达载体pGEX-6P-1-TMEM39A与GenBank中的TMEM39A(登录号:BC021277.2)的核苷酸序列同源性为99.9%,氨基酸序列同源性为100%。重组蛋白GST-TMEM39A在69和60 ku处出现两条特异性条带,采用单因素方法对不同温度、IPTG浓度、A600nm值及时间进行分析得出GST-TMEM39A的最佳诱导条件为25℃、A600nm值为0.6~0.8、IPTG浓度为0.1 mmol/L诱导9 h;在此基础上进行大量表达,并以可溶性形式获得了TMEM39A与GST蛋白的融合表达。结论成功构建了TMEM39A的原核表达载体,确定了GST-TMEM39A的最佳表达条件并实现其的可溶性表达,为后续纯化TMEM39A、制备抗体开展功能研究奠定物质基础,并为深入探讨TMEM39A与许多疾病或病毒的关系提供科学依据。

Objective To construct prokaryotic expression vector of human TMEM39A,optimize expression conditions and obtain soluble TMEM39 A.Methods TMEM39A was amplified from HEK293 cells by RT-PCR to construct its prokaryotic expression vector pGEX-6 P-1-TMEM39A,and the induced expression was conducted at different temperatures,IPTG concentrations,A600 nm values and time conditions.Finally,expression of the recombinant protein GST-TMEM39 A was carried out to analyze its solubility and antigenicity by using the best induction conditions.Results The nucleotide sequence homology of the recombinant expression vector pGEX-6 P-1-TMEM39A and TMEM39 A in GenBank(BC021277.2)was 99.9%and the amino acid sequence homology was 100%.The recom-binant protein GST-TMEM39 A showed two specific bands at 69 ku and 60 ku by Western blot analysis.The optimal induction conditions for GST-TMEM39 A was 25℃,A600 nm value of 0.6-0.8,0.1 mmol/L IPTG induced 9 h.On this basis,the fusion expression of TMEM39 A and GST protein was obtained in a soluble form.Conclusions The prokaryotic expression vector of TMEM39 A is successfully constructed,the optimal expression condition of GST-TMEM39 A is determined and its soluble expression is realized,which lays a physical foundation for the subsequent purification of TMEM39 A and the preparation of antibodies for functional studies,and provides scientific basis for further study about the relationship between TMEM39 A and many diseases or viruses.