CCK39／UreB双基因融合表达载体的构建及其在大肠杆菌中的表达

Construction and expression of the fusion gene CCK39/UreB in recombinant Escherichial coli BL-21(DE3)

本研究报道了猪源性胆囊收缩素39肽(Cholecystokinin 39,CCK39)和猪肠道大肠菌群脲酶B亚基(Urease B,UreB)融合的原核表达载体的构建,以及兼有CCK39和UreB功能活性域的融合蛋白的表达。利用RT-PCR从猪十二指肠总RNA中克隆CCK39,再通过PCR从猪肠道产脲酶大肠菌群质粒中克隆UreB,之后将扩增所得的两基因先后定向插入原核表达载体pET43a(+)中,构建双基因融合表达载体pET43a(+)/CCK39/UreB,并转化至大肠杆菌(Escherichia coli)BL-21(DE3)中。重组质粒pET43a(+)/CCK39/UreB经PCR、双酶切鉴定和序列分析,证实已成功构建双基因融合表达载体。重组表达菌经isopropylthio-β-D-galactoside(IPTG)诱导表达了分子量约为80kD的融合蛋白,与预期分子量相同,主要以可溶蛋白的形式存在于细胞质中;用Ni2+-NTA对可溶蛋白进行亲和纯化,纯度大于95%;Western blotting分析结果显示重组融合蛋白可分别为CCK8和UreB抗血清识别,具有良好的反应原性。本研究所表达的融合蛋白为研制抗CCK/Urease双效疫苗奠定了基础。

The aims of this research were to construct prokaryotic expression vector containing fusion gene of Cholecystokinin 39 （CCK39） of pig and Urease subunit B （UreB） of coliform bacteria, and then to express the fusion protein in reconbinant Escherichia coli BL21（DE3）. The CCK39 gene was amplified by RT-PCR from the extracted total RNA of pig＇s duodenum, and the UreB gene was then amplified by PCR from the extracted plasmid DNA of bacillus of coliform bacteria from pig＇s intestinal content. Then the CCK39 and the UreB were inserted into the prokaryotic expression vector pET43a（＋） to construct a recombinant fusion expression vector pET43a（＋）/CCK39/UreB and then, the recombinant vector was identified by PCR, endonuclease digestion and sequence analysis. It was identified that the gene fragment of CCK39 at length of 117 bp and UreB at length of 324 bp were amplified and cloned into the vector pET43a（＋） successfully. The recombinant vector was transformed into Escherichia coli BL21（DE3） and induced the expression of CCK39/UreB fusion protein with a molecular mass of approximately 80 kD by using isopropylthio-β-D-galactoside （IPTG） as inducer. The fusion protein was mostly located in the cytoplasm and it was soluble. The soluble protein was collected and purified by Ni^2＋-NTA column chromatograph and then reached a purity of more than 95%. It was proved by western blotting that the fusion protein could react with rabbit anti-CCK8 antiserum and rabbit anti-UreB antiserum. Therefore, the expressed fusion protein has good antigenicity. This work established a good foundation for further study on the production of anti-CCK/Urease vaccines.