【作者】 汪春义；

【导师】 盛军；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 目前,生产人胰岛素大多采用人工合成、化学提取和基因工程等方法。基因工程生产人胰岛素一般用大肠杆菌表达,且表达的胰岛素是以胰岛素原的形式存在。它必需经过后序处理,才具有生物活性。而且它的使用大多以注射为主,既麻烦又给病人带来一定的痛苦。随着生物技术的发展,对天然药物研究的开发和利用越来越得到人们的重视。本研究首次将构建了霍乱毒素B亚单位(CTB)与人胰岛素融合蛋白基因的植物表达载体通过农杆菌感染而将其整合到了人参愈伤组织细胞的染色体上,结果表明人胰岛素基因在人参愈伤组织细胞内得以表达。从小鼠实验看出,人参细胞亦有降血糖作用,而转化了人胰岛素基因的人参细胞的降血糖作用更明显,与非转化的人参细胞相比具有显著性差异。但小鼠的甘油三酯和胆固醇下降不明显。本研究设计独特,充分发挥了霍乱毒素B亚单位(CTB)的肠道吸附作用与人胰岛素的治疗作用,并将它们通过人参细胞表达,使得现代生物技术与我国的传统中药有机地结合在一起,为今后胰岛素的口服用药开辟了一条新路。更多还原

【Abstract】 Diabetes is a kind of chronic disease ,the statistic data indicates that the quantities of diabetes suffers have the increasing tendency year by year ,and now injecting insulin is the most effective method for prevention of diabetes. At present , the production of insulin mostly use the methods of synthesis , chemical extraction and genetic engineering and so on , and insulin is usually expressed by Eco.li . But the expression product is pre-insulin , which has no activity , it only can be used after being handled . In the same time, injection brings the pain to the patients . This research goal lies in that we can construct CTB-human insulin fusion gene plant expression vector,and introduce fusion gene into cell chromosome in ginseng cell line throughAgrobacterium–mediated transformation system ,so that we can establish ginseng cell lines expressing CTB-human insulin fusion proteins for oral medication in future .First ,we select ginseng cell biased codons according to the cDNA sequence of human pre-insulin signal peptide (SP) ,chain B ,chain A , chain C and CTB gene provided by Gene Bank , transform human pre-insulin (SPBCA) and CTB separately ,and design Furin proteinase digestion position spots on two ends of chain C and three GP peptides on C end of chain C . SPBCA gene and CTB gene are amplied by PCR method ,after that BCA gene and BA gene are also amplied with the template SPBCA . Four fragments of purpose genes are sub-coloed into the vectors pMD18-T to construct the vectors pMSPBCA , pMBCA ,pMBA, pMCTB. The colone vectors are digested by corresponding enzyme ,and the goal gene fragments SPBCA ,BCA ,BA and vector fragment pMD-CTB are recycled , then they are constructed into fusion clone vectors pMCSPBCA , pMCBCA , pMCBA . In the same , these fusion clone vectors are digested by BamH I and Sac I , the fusion gene fragments CTB-SPBCA , CTB-BCA , CTB-BA are recycled , and then they are separtely cloned to real nuclear expression vector pBI121 to become plant expression vectors pBCSPBCA , pBCBCA , pBCBA . Recombinant plasmids are extracted to be transformed into Agrobacterium LBA4404 , the fusion genes are separtely transinfected and joined into ginseng cell line through Agrobacterium–mediated transformation method . Their cell genome DNA is extracted after screening positive cell strains with 67-V medium including 50mg/L of G418 , then the fusion genes are detected if they have been introduced into the chromosome of ginseng cells by PCR method . In the same , their cell total mRNA is also extracted to prove their existence of duplication products for fusion genes through RT-PCR method and agarose gel electrophoresis . Western-blotting method and ELISA method are used to detect goal fusion proteins and their protein contents .In addition to , non-transformed ginseng cells , transformed ginseng cells and sterile physiological saline are respectively injected into mouse abdominal cavity and administrated orally to examine the changes of blood sugar ,the triglyceride and the total cholesterol density after injection , as well as blood sugar density change after oral administration .The experimental results show that CTB-SPBCA , CTB-BCA , CTB-BA fusion genes have successfully been introduced into the ginseng cells , it has been identified by enzyme digestion reaction of PCR products with screened transgenic cell genome DNA as the template . Total mRNA of transgenic cell is extracted , it has been proved by RT-PCR reaction and the agarose gel electrophoresis that duplication products of CTB-SPBCA , CTB-BCA , CTB-BA genes are existent . Western-blotting examination result shows that the special protein bands appear at these positions whose relative molecular weight are 25KD , 23KD and 19KD , this demonstrates that three kinds of fusion proteins can specially combine with human insulin antibody . ELISA method test shows that goal proteins have been expressed , Protein contents of CTB-SPBCA , CTB-BCA , CTB-BA are respectively 6.03uIU/ml , 6.81uIU/ml , 5.31uIU/ml . The mouse blood sugar examination results indicate that ginseng cell can decrease the content of blood sugar , transformed ginseng cell is more remarkable in falling the content of blood sugar , but the contents of triglyceride and the total cholesterol change little . non-transformed ginseng cell supernate , transformed ginseng cell supernate and steril physiological saline are respectively injected into mouse abdominal cavity and administrated orally to examine the changes of blood sugar ,the triglyceride and the total cholesterol density around injection , as well as blood sugar density change around oral administration . After the results are handled by SPSS13.0 statistics software ,the repetition survey variance analysis finds that three group blood sugar values after injection have obvious difference with the values before injection (the P values are 0.000) . The values of blood sugar all decrease in non-transformed ginseng cell group and transformed ginseng cell group ,but its value of blood sugar is high in blank control group . The value of blank control group has obvious difference with that of non-transformed ginseng cell group and transformed ginseng cell group( the P value are 0.000) , and it is the same for non-transformed ginseng cell group and transformed ginseng cell group(the P value is 0.019) , transformed ginseng cell group is more remarakable in the function of falling blood sugar .But the contents of triglyceride and total cholesterol between transformed group and non-transformed group have no significant difference . The values of blood sugar all decrease in non-transformed ginseng cell group and transformed ginseng cell group on 2 hours and 6 hours after oral administration compared with pre-administration, the difference is remarkable (P<0.05) , but its value of blood sugar changes a little in blank control group , there is not notable difference between pre-administration and post-administration . transformed ginseng cell group is obviously different from non-transformed ginseng cell group , transformed ginseng cell group is more remarakable in the function of falling blood sugar . The diference of blood sugar values are not remarkable on 24 hours after oral administration .In a word , this study has successfully constructed plant expression vectors pBCSPBCA , pBCBCA and pBCBA containning CTB-insulin fusion genes , and obstained transgenic ginseng cell line that can express CTB-insulin fusion proteins through transformation method mediated by Agrobacterium , it has also been proved by animal test . These have laid a good foundation for the development and application of oral administration insulin preparation , and also opened a new road for the development of natural medicine .更多还原