人表皮角质形成细胞桥粒芯糖蛋白4胞外区域基因片段的克隆

Cloning the gene fragments of desmoglein 4 extracellular domains from human keratinocytes

目的:扩增人表皮角质形成细胞桥粒芯糖蛋白4胞外区域EC1,EC2,EC3和EC4的核酸序列。方法:实验于2004-11/2005-09在四川大学华西医学中心感染免疫研究室完成。通过反转录聚合酶链反应法从人正常皮肤组织表皮角质形成细胞中抽提RNA,反转录合成cDNA,聚合酶链反应扩增表皮角质形成细胞桥粒芯糖蛋白4胞外区域EC1,EC2,EC3和EC4目的基因;应用基因重组技术将目的基因分别与质粒pET32a在T4DNA连接酶作用下相连接,转化E.coliDH5a感受态细菌,用含氨苄青霉素的LB培养基平板筛选转化菌,阳性重组子DNA序列测定鉴定。结果:反转录聚合酶链反应法扩增产物经凝胶电泳得到均约为350bp的4条条带;质粒载体pET32a连接的目的基因经序列测定后与在Genbank登录的桥粒芯糖蛋白4胞外区域EC1,EC2,EC3和EC4核酸序列完全一致,4个桥粒芯糖蛋白4胞外区域cDNA读码框架正确。结论:成功克隆得到的桥粒芯糖蛋白4胞外区域EC1,EC2,EC3和EC4多肽片断。

AIM： To amplify the nucleotide sequence of desmoglein 4 extracellular domains （EC1, EC2,EC3 and EC4） from human keratinocytes. METHODS： The experiment was conducted at the Department of Infection ＆ Immunity, West China Center of Medical Sciences, Sichuan University from November 2004 to September 2005. RNA was obtained from norreal human keratinocytes through reverse transcriptlon-polymerase chain reaction （RT-PCR） and then cDNA was synthesized. Target genes of desmoglein 4 extracellular domains（EC1,EC2,EC3 and EC4） were amplified by polymerase chain reaction. With the technique of gene recombination, target genes were linkaged with pET32a plasmids respectively by T4DNA ligase, which translated into E.coli DH5a competent germs. Screening of translated germs was done by LB medium with Ampicillin. DNA sequences of positive recons were identified finally. RESULTS： Four bands with all the length of 350 bp were obtained by RT-PCR. Consequently, four expressed plasmids of desmoglein 4 extracellular domains （EC1,EC2,EC3 and EC4） were.constructed, and sequencing proved that the gene sequences and read frames were correct. CONCLUSION： Desmoglein 4 extracellular domains （EC1,EC2,EC3 and EC4） polypeptide segments were successfully cloned.