抗药性相关糜蛋白酶基因真核表达载体的构建和鉴定

目的：构建抗药性相关基因真核表达载体。方法：采用PCR方法，以淡色库蚊抗药性品系cDNA文库为模板，扩增出抗药性品系中高表达糜蛋白酶(chymotrypsin)基因，经T／A克隆测序鉴定后，扩增、纯化质粒，经双酶切后纯化目的片段，克隆到真核荧光表达载体pEGFP—C3中。结果：PCR和酶切鉴定表明，构建成功真核荧光表达载体pEGEP—C3／chymotrypsino结论：PCR加双酶切鉴定的方法构建表达质粒，迅速省时，结果可靠。

Construction and Identification of Eukaryotic Expression Plasmid of Resistance-related Chymotrypsin Gene

objective: To construct the expression plasmid of resistance-related chymotrypsin gene. Methods: The methods of PCR and double enzymes cutting were used. The complete chymotrypsin gene was obtained from the cDNA library of the resistance strain of Culex pipiens pollens. Identified by sequencing the plasimid of the T/A cloning, the fragment of chymotrypsin gene was cut down from the pGEM-T easy vector that contained the gene, and then inserted to the eukarytic expression vector pEGFP-C3. Results: Identified by PCR and enzyme digest, the expression plasmid pEGFP-C3/chymotrypsin was constructed exactly. Conclusion: It is very convenient to construct and identify the expression plasmid of resistance-related chymotrypsin gene by means of PCR and enzyme cutting, and the result is credible.