棘阿米巴原虫actin1基因原核表达载体构建与鉴定

Construction and identification of actin1 prokaryotic expression vector of Acanthamoeba spp

棘阿米巴角膜炎的临床诊断比较困难,以角膜刮片的病原学诊断为主,样本采集困难,临床诊断率低。本研究构建了棘阿米巴原虫actin1基因进行原核表达载体,并进行鉴定。以棘阿米巴滋养体cDNA为模板,通过特异性引物,经聚合酶链反应,获得actin1基因开放阅读框（ORF）片段,进行克隆及序列分析,经双酶切,连接,构建重组表达载体pET22b（＋）-actin1。将重组载体转化入大肠杆菌（E.coli）DH5α,筛选出阳性菌落,经PCR扩增和酶切鉴定后,阳性质粒送样测序;提取阳性质粒,转化入E.coli BL21（DE＋）,1mol/L IPTG诱导重组蛋白r-actin1的表达。自棘阿米巴滋养体cDNA扩增得到约774bp的actin1基因片段;经PCR和酶切鉴定表明,重组表达载体pET22b（＋）-actin1构建成功;重组菌株BL21（DE＋）/pET22b（＋）-actin1,经培养、诱导、SDS-PAGE电泳,出现1条相对分子质量约为30 000重组蛋白条带,r-actin1表达诱导表达成功。成功构建重组表达载体pET22b（＋）-actin1,并诱导表达。

To construct prokaryotic expression vector of Acanthamoeba spps actin1 gene,and analyze its expression,the open reading frame（ORF）of actin1 gene was amplified with specific primers by polymerase chain reaction（PCR）using the cDNA template of Acanthamoeba spp.The PCR product was cloned into prokaryotic expression vector pET22 b（＋）,digested with restriction enzymes HindⅢand NdeⅠ.The recombinant vector pET22 b（＋）-actin1 was transformed into E.coli DH5α,and the positive clone were then selected and identificated by PCR analysis.The recombinant plasmids were extracted and digested by restriction enzymes NdeⅠ and HindⅢ.The positive plasmid was transformed into E.coli BL21（DE3）and induced with 1 MIPTG for expression of recombinant protein actin1（ractin1）.774 bp actin1 gene segment was amplified by PCR.PCR and restriction enzyme digestion demonstrated the construction of recombinant plasmid pET22 b（＋）-actin1.Recombinant protein ractin1 was successfully expressed by the recombinant strain BL21（DE＋）/pET22 b（＋）-actin1.The SDS-PAGE results showed a protein band at 30 000.The study successfully constructed the recombinant expression vector pET22 b（＋）-actin1 and induced ractin1 expression.