基于重组PCR法构建人骨保护素N端的酵母表达载体  

Construction of Pichia Expression Vector Encoding N-terminal Fragment of Osteoprotegerin Using Recombinant PCR

作　　者：李晓霞[1] 朱志峰[2] 王宝利[2] 戴芳[2] 郭刚[2] 张镜宇[2] 

机构地区：[1]天津医科大学微生物教研室,300070 [2]天津医科大学代谢病医院内分泌研究所

出　　处：《天津医药》2006年第11期753-755,共3页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(项目编号:30300171);天津市自然科学基金资助项目(项目编号:043606811)

摘　　要：目的:构建骨保护素(OPG)N端片段的酵母表达载体。方法:OPGN端D1-D4域仅由2个外显子编码。以人基因组DNA为模板,PCR分别扩增外显子2和外显子3,并使外显子2的3′引物和外显子3的5′引物具有相互重叠的一段序列。以2种PCR产物混合物为模板,采用重组PCR扩增OPGN端编码序列,拟插入酵母分泌型表达载体pPIC9K。为使未来分泌表达产物具有完全正确的N末端,再次采用重组PCR策略,将pPIC9K信号肽裂解位点前的一段序列(含单一限制酶位点BamHⅠ)与OPGN端编码序列拼接在一起,从BamHⅠ和EcoRⅠ位点插入pPIC9K。结果:得到了OPGN端酵母表达载体,测序证实插入序列正确。结论:重组载体的获得为基因工程研制具有生物活性的OPGN端片段奠定了基础。Objective： To construct pichia expression vector encoding N-terminal fragment of osteoprotegerin （OPG）. Methods： The bioactivity of OPG is dependent on the presence of the N-terminal D1-D4 domain. This region is encoded by only two exons, exon 2 and exon 3. In the present study, two exons of the sequence coding were amplified respectively and then linked using recombinant PCR method. As designed, the antisense primer for exon 2 has a sequence of 20 bp that is homogeneous with the sense primer for exon 3. The PCR products for exon 2 and 3 were mixed as template. The sense primer for exon 2 and antisense primer for exon 3 were used together to initiate the synthesis of the coding sequence for D1-D4 region. The secretory pichia expression vector pPIC9K was selected for the expression of the target protein. To ensure the expressed protein has authentic N-terminal sequences after the removal of the signal peptide by endogenous pretease KEX2, a sequence in pPIC9K just before the cleavage site of KEX2 involving a unique BamH Ⅰ site was connected with the sequence encoding D1-D4 region through another recombinant PCR. This chimeric molecule was then cloned into pPIC9K between the sites of BamH Ⅰ and EcoR Ⅰ. Results： The recombinant vector was obtained with the correct sequence in the insert. Conclusion： The construction of this recombinant molecule provides a sound basis for the production of bioactive N-terminal fragment of osteopretegerin by genetic engineering.

关 键 词：破骨细胞 成骨细胞 聚合酶链反应 克隆 分子DNA 重组 

分 类 号：R373.21[医药卫生—病原生物学]