人跨膜蛋白39A原核表达载体构建、条件优化及可溶性表达

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。

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

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.