在DH5α中拼接构建ADAM10全长真核表达载体的基因突变

Splicing and Construction of a Eukaryotic Expression Vector Containing Full Length ADAM10 Gene and Its Mutation in E.coli DH5α

目的:构建ADAM10真核表达载体,为进一步研究其生物学功能打基础。方法:将人ADAM10的上下两部分基因片段(分别为全长基因的1～910bp和911～2 247bp片段),依次与真核表达载体pcDNA3.1相连,以大肠杆菌DH5α或BL21(DE3)作为感受态宿主菌用于转化连接产物,拼接成全长的阳性克隆通过PCR、酶切和测序鉴定。结果:ADAM10下段基因与已正确连入上段的pcDNA3.1重组质粒拼接时,若用DH5α为感受态菌,则下半段出现碱基插入增加512bp,测序结果显示为ADAM10基因第1 531 bp～2 042 bp间的序列有紧邻的双份;若用BL21(DE3)为感受态,则无突变。结论:将ADAM10基因与pcDNA3.1真核表达载体依次拼接构建重组质粒时,以DH5α为宿主菌可出现基因序列增加的罕见突变,而以BL21(DE3)为宿主则无突变,由此成功构建ADAM10全长基因与pcDNA3.1的重组质粒。

Objective:To splice and construct a eukaryotic expression vector containing full length ADAM10 gene.Method:The ADAM10(up) sequence(from 1nt to 910nt) and ADAM10(down) sequence(from 911nt to 2 247nt) were spliced and subcloned into eukaryotic expression vector pcDNA3.1,the recombinant plasmid were transformed into E.coli DH5α and BL21(DE3) respectively,then were identified by PCR,digestion with restriction enzyme and sequencing assay.Result:When the splicing and construction were performed in E.coli DH5α,insertion mutation was observed in the recombinant plasmid by increasing 512bp(the fragment from 1 531nt to 2 042nt of ADAM10 was replicated and inserted into the recombinant plasmid);and when the operation was performed in E.coli BL21(DE3),no mutation in the recombinant plasmid.Conclusion:During splicing and construction of a eukaryotic expression vector containing full length ADAM10 gene,insertion mutation could occur in recombinant plasmid by using E.coli DH5α competence,and no mutation was detected by using E.coli BL21(DE3) competence,then a recombinant plasmid containing full length ADAM10 gene was constructed successfully,which provides a basis for the study of biological effects of ADAM10.