[Construction and expression of the fusion gene CCK39/UreB in recombinant Escherichia coli BL-21(DE3)].
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 recombinant 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-beta-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 Ni2+-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.
College of Food Science, South China Agricultural University, Guangzhou 510640, China.
Weiyang
W
Zhang
Yan
Y
Li
Tongshan
T
Wu
Wenhua
W
Luo
Bin
B
Hu
Wenfeng
W
Hu
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D050939:Gene Fusion; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D013552:Swine
D001426:Bacterial Proteins; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C090404:UreE protein, Bacteria; C032740:cholecystokinin 39; D002766:Cholecystokinin