空肠弯曲菌cdtA、cdtB及cdtC真核表达重组载体的构建与表达

Construction and expression of eukaryotic expression recombinant vector of campylobacter jejuni cdtA、cdtB、cdtC gene

目的以空肠弯曲菌(CJ)细胞扩张毒素(cytolethal distending toxi,CDT)cdtA、cdtB及cdtC为目的基因,构建CJ-cdtA、cdtB及cdtC基因的真核表达重组载体pcDNA3.1(+)-cdtA,pcDNA3.1(+)-cdtB,pcDNA3.1(+)-cdtC,为CJ核酸疫苗的研制提供实验材料。方法用Primer5.0软件分析GenBank中空肠弯曲菌细胞扩张毒素cdtA、cdtB及cdtC基因序列,设计合成相应特异性引物,引入EcoRI、BamHⅠ和XhoⅠ酶切位点,PCR扩增cdtA、cdtB及cdtC目的基因片段,定向插入真核表达载体pcDNA3.1(+)多克隆酶切位点中,构建重组体pcDNA3.1(+)-cdtA、pcDNA3.1(+)-cdtB及pcDNA3.1(+)-cdtC;通过酶切分析、PCR鉴定及测序鉴定,筛选阳性重组体。结果扩增出特异的cdtA、cdtB及cdtC基因片段,大小约为807bp、798bp、570bp;双酶切及测序鉴定证明成功构建了CJ真核表达重组载体pcDNA3.1(+)-cdtA、pcDNA3.1(+)-cdtB及pcDNA3.1(+)-cdtC。结论 PCR扩增得到了大小约为807bp、798bp、570bp的CJ-cdtA、cdtB及cdtC目的基因片段;并成功构建了pcDNA3.1(+)-cdtA、cdtB及cdtC真核表达重组载体。

Objective The recombinant plasmid containing the cytolethal distending toxin （CDT） gene of campylobacter jejuni （CJ） was constructed to provide a foundation for future development of C. jejuni DNA vaccine.Methods Primers were designed by Primer 5. 0 software according to CJ-cdtA, cdtB, cdtC gene sequences provided from GenBank. Polymerase chain reaction （PCR） was used to amplify the cdtA, cdtB, cdtC gene . The fragment was subcloned into appropriate site of pcDNA3. 1 （ ＋ ） eukaryotic expression vector by restriction enzyme digestion and linking reactions. The positive recombinant was identified by restriction endonuclease digestion , PCR amplification and sequencing. Results The 807bp, 798bp, 570bp-cdtA, cdtB, cdtC specific gene fragments were amplified. Restriction enzyme analysis and sequencing showed that the eukaryotic expression recombinant pcDNA3. 1 （ ＋ ）-cdtA, cdtB, cdtC were successfully constructed. Conclusion The cdtA, cdtB, cdtC gene fragments are successfully amplified , and the eukaryotic expression recombinant pcDNA3. 1 （ ＋ ）-cdtA, cdtB, cdtC are successfully constructed .