降钙素基因在乳酸杆菌中的克隆

Cloning of Calcitonin Gene in Lactobacillus

根据鲑鱼降钙素基因的序列,合成了鲑降钙素基因。首先将该基因连接到以ThyA基因为选择压力的非抗性表达载体pW425t中,转化ThyA基因缺陷的大肠杆菌X51。用SacI和KpnI双酶切法和PCR法筛选出阳性重组质粒,然后采用电穿孔法将阳性重组质粒pWCT转化到ThyA基因缺陷的乳酸杆菌中。用PCR法筛选阳性重组质粒,并进行序列测定。结果表明,目的基因所编码的氨基酸序列与天然鲑降钙素的氨基酸序列完全一致。我们得到了含降钙素基因的重组乳酸杆菌。

Calcitonin gene was synthesized according to the sequence of salmon Calcitonin gene. The gene andunantibitic expressive vector pW425t were both digested by SacI and KpnI. Two parts were connected by T4 DNAligase. The recombined plasmid pWCT proved containing Calcitonin gene by restriction enzyme digestion andPCR. 100bp fragment was amplified by PCR from the recombinant plasmid pWCT using the primers P1/P2. Therecombinant plasmid pWCT was then cloned into Lactobacillus by electric-translation.The sequence of the Calci-tonin gene was determined. Compared with those of salmon Calcitonin, the identities of predicted amino acids are100%. The recombined Lactobacillus will be used to express salmon Calcitonin, and be used to prevent and treatCaged Layer Osteoporosis.