CAJ | 학술논문

目的开发激肽释放酶基因工程产品,为开展基因治疗高血压奠定基础.方法提取人胰腺组织总RNA,逆转录后PCR扩增激肽释放酶cDNA.回收、补平后插入质粒KS,构建出中间载体KSKK,酶切鉴定后双向测序分析激肽释放酶基因序列.从KSKK中切出激肽释放酶原及激肽释放酶基因,插入真核表达载体PET-28b(+),经酶切鉴定后,进行核苷酸序列分析和融合蛋白表达.结果本实验克隆的激肽释放酶基因与GenBank报告的激肽释放酶基因相比,有一个碱基不同,同源性为99.8%.将IPTG诱导表达进行SDS-PAGE电泳,与蛋白标准品比较在31800处可见明显的高表达带.免疫印迹实验表明重组蛋白具有KK的抗原性.结论已成功克隆并表达了人组织激肽释放酶基因,为进一步开发基因工程产品及进行基因治疗高血压研究奠定了基础.
목적개발격태석방매기인공정산품,위개전기인치료고혈압전정기출.방법제취인이선조직총RNA,역전록후PCR확증격태석방매cDNA.회수、보평후삽입질립KS,구건출중간재체KSKK,매절감정후쌍향측서분석격태석방매기인서렬.종KSKK중절출격태석방매원급격태석방매기인,삽입진핵표체재체PET-28b(+),경매절감정후,진행핵감산서렬분석화융합단백표체.결과본실험극륭적격태석방매기인여GenBank보고적격태석방매기인상비,유일개감기불동,동원성위99.8%.장IPTG유도표체진행SDS-PAGE전영,여단백표준품비교재31800처가견명현적고표체대.면역인적실험표명중조단백구유KK적항원성.결론이성공극륭병표체료인조직격태석방매기인,위진일보개발기인공정산품급진행기인치료고혈압연구전정료기출.
[Objective] To develop recombinant human pancreatic kallikrein and lay a foundation of hypertension. [Methods] Total RNA was extracted from human pancreas and then human tissue kallikrein gene cDNA was amplified by reverse-transcription PCR and cloned to the KS plasmid. Then amplify the mature kallikrein gene from pBluescripe Ⅱ KSKK(+)and insert into pET28b(+). The clones were identified by enzyme digestion and sequenced,and fusion protein was expression under the instruction of IPTG. [Results] SDS-PAGE profile showed a clear protein band with a relative molecular weight of 31 800. Western blot proved that the expressed protein showed specific antigenicity to human serum kallikrein. [Conclusion] Human pancreatic kallikrein gene was successfully cloned and expressed. It laid a foundation of further study in gene therapy for treating hypertensive diseases and other renal diseases or be used in other researchs.