中国人胚胎肝组织来源纤溶酶原kringle5基因的克隆与序列分析  

Cloning and sequencing of plasminogen kringle5 cDNA derived from Chinese embryonic hepatocytes

作　　者：甘舜进[1] 胡朝晖[2] 曾征宇[2] 燕启江[2] 陈广南[2] 

机构地区：[1]北京潞河医院心内科,北京市101149 [2]广州医学院金域医学检验中心,广东省广州市510182

出　　处：《中国组织工程研究与临床康复》2007年第38期7655-7657,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:分离、克隆和测定中国人纤溶酶原Kringle5功能区基因,为进一步研究其功能奠定基础。方法:实验于2002-06/2003-05在广州医学院金域医学检验中心完成。①实验材料:国人胚肝组织取自广州医学院第一附属医院的流产胚胎(取得家属同意,并经广州医学院第一附属医院伦理委员会批准)。pET21a(+)载体购自Novagen公司,大肠杆菌BL21(DE3)为医学检验中心保存。引物均由上海生工合成。②实验方法:从国人胚肝组织中提取mRNA,用反转录-聚合酶链反应方法将人纤溶酶原Kringle5的cDNA扩增出来,克隆到pET21a(+)载体中测序。③实验评估:采用紫外分光光度仪和琼脂糖凝胶电泳分析胚肝组织总RNA的抽提结果;经琼脂糖凝胶电泳鉴定Kringle5的反转录-聚合酶链反应扩增结果;pET-Kringle5重组质粒的酶切鉴定;序列测定。结果:①胚肝组织提取总RNA结果:提取的总RNA经紫外分光光度仪测得A260nm/A280nm>1.8,A260nm/A270nm>1.2,表明无蛋白残留;电泳结果显示提取的总RNA有明显的28S、18S两条带,说明RNA基本完整。②Kringle5的反转录-聚合酶链反应扩增结果:人Kringle5cDNA片段长为240bp,加上引物设计的2个酶切位点,总长度为258bp,聚合酶链反应产物长度与该长度一致,符合预期结果。③pET-Kringle5重组质粒的构建和酶切鉴定结果:用引物所带的限制性内切酶BamHⅠ、NdeⅠ双酶切,结果有250bp左右条带出现。④序列测定结果:证实国人纤溶酶原Kringle5功能区基因被成功克隆,序列分析证实为该基因,未发现有基因突变或多态性现象,但第153位核苷酸与文献比较存在碱基替代现象,其组成的密码子由于遗传的简并性,所编码的氨基酸相同,并未造成氨基酸组成的改变。结论:中国人纤溶酶原Kringle5功能区cDNA基因编码序列与国外文献报道的相应序列可能存在碱基替代现象。AIM： To lay foundation for further study on the function of plasminogen Kringle5, the plasminogen kringle 5 cDNA was isolated, cloned and sequenced. METHODS： The experiment was conducted in the Center of Medical Laboratory of Guangzhou Medical College between June 2002 and May 2003. The total mRNA was isolated from Chinese embryonic hepatocytes （the fetus of abortion from First Affiliated Hospital of Guangzhou Medical College; the informed consent was obtained from the family and permission from Hospital Ethics Committee）. The plasminogen Kringle5 cDNA was synthesized and amplified from the mRNA by RT-PCR and the PCR product was cloned into pET21a （＋） vector （Novagen Company） and subjected to DNA sequencing. Ultraviolet spectrophotometer and agarose gel electrophoresis （AGE） were used to detect the result of total RNA extract; the amplification of Kringle5 by RT-PCR was identified by AGE; pET-Kringle5 recombinant plasmid was enzyme digested and sequenced. RESULTS; （1）The total RNA was successfully isolated from Chinese fetus hepatocytes, and A260nm/A80nm〉1.8,A60nm,A270nm〉1.2 indicated no protein remained; AGE results showed that there were obvious two bands of 28 S and 18 S, indicating that RNA was basically complete. （2）The fragment of plasminogen Kdngle5 cDNA was 240 bp, and 258 bp by adding 2 enzyme digestion sites. It was the same to the length of RT-PCR products, which was in accordance with prediction. （3） The recombinant pET-Kringle5 vector had been successfully constructed. Enzyme digestion by Nde l and BamH l presented a 258 bp fragment. （4）The plasminogen kingle5 cDNA was successfully cloned and the DNA sequencing analysis showed that no mutation or polymorphism was found, except a silent base exchange at 153 base, without changes of amino of the plasminogen Kdngle5. CONCLUSION： The coding sequence of Chinese plasminogen Kringle5 cDNA may have a silent base exchange compared with the reported sequence derived from foreigner.

关 键 词：纤维蛋白溶酶原 克隆 生物 序列分析 组织构建 

分 类 号：R333.4[医药卫生—人体生理学]