建立甲胎蛋白的纳米抗体-rt-pcr超灵敏分析方法-to establish a method for ultra-sensitive analysis of alpha fetoprotein by nano-antibody rt - pcr..docxVIP

下载本文档

4
0
约7.76万字
约 100页
2018-05-28 发布于上海
举报
版权申诉

建立甲胎蛋白的纳米抗体-rt-pcr超灵敏分析方法-to establish a method for ultra-sensitive analysis of alpha fetoprotein by nano-antibody rt - pcr..docx

1、有哪些信誉好的足球投注网站（book118）网站文档一经付费（服务费），不意味着购买了该文档的版权，仅供个人/单位学习、研究之用，不得用于商业用途，未经授权，严禁复制、发行、汇编、翻译或者网络传播等，侵权必究。。
2、本站所有内容均由合作方或网友上传，本站不对文档的完整性、权威性及其观点立场正确性做任何保证或承诺！文档内容仅供研究参考，付费前请自行鉴别。如您付费，意味着您自己接受本站规则且自行承担风险，本站不退款、不进行额外附加服务；查看《如何避免下载的几个坑》。如果您已付费下载过本站文档，您可以点击这里二次下载。
3、如文档侵犯商业秘密、侵犯著作权、侵犯人身权等，请点击“版权申诉”（推荐），也可以打举报电话：400-050-0827(电话支持时间：9:00-18:30)。
4、该文档为VIP文档，如果想要下载，成为VIP会员后，下载免费。
5、成为VIP后，下载本文档将扣除1次下载权益。下载后，不支持退款、换文档。如有疑问请联系我们。
6、成为VIP后，您将拥有八大权益，权益包括：VIP文档下载权益、阅读免打扰、文档格式转换、高级专利检索、专属身份标志、高级客服、多端互通、版权登记。
7、VIP文档为合作方或网友上传，每下载1次，网站将根据用户上传文档的质量评分、类型等，对文档贡献者给予高额补贴、流量扶持。如果你也想贡献VIP文档。上传文档

建立甲胎蛋白的纳米抗体-rt-pcr超灵敏分析方法-to establish a method for ultra-sensitive analysis of alpha fetoprotein by nano-antibody rt - pcr.

AbstractThere is a heavy chain antibody with lack of natural light chain in camel source . Variable domain of heavy chain of heavy-chain antibody is consist of the heavy chain variable region (variable region) genetic engineering antibody, which is called nanobody because of its small weight. Compared with ordinary antibodies and recombination single antibody, nanobody has many advantages such as small molecular weight, water-soluble high, stable strong, easy to express etc. Therefore, it is applied in many fields.A fetoprotein (AFP) is mainly composed in the fetal liver that molecular weight is 69 KDa. AFP in healthy adult serum is below 30 ng/mL. However, AFP level apparently increase in serum when hepatocyte undergoes a cancer change. Since AFP is a specific clinical diagnosis of primary liver cancer. In this research,we construct sandwich Enzyme-linked immunosorbent assay to detect AFP by using nanobody that specifically bind to AFP. The main research contents are as follows:The establishment of the AFP immune library After Alpacas immunized four times with AFP, alpacas’s serum was collected two weeks later and isolated hemameba. mRNA was extracted from hemameba. cDNA library was constructed by reverse PCR.We obtained VHH genes by nest-PCR. Using restriction enzyme NotⅠand SfiⅠ,VHH genes were inserted into pHENⅠ Vector. The recombination plasmids were transferred into E.coliTG1 by electrotransformation.Biopanning from immune Library Phages that specifically bind to AFP were obtained by Solid-phase biopanning, reducing AFP concentration in each round with alternate using BSA and OVA blocking agent. After three rounds selection, 95 clones were picked to bind AFP. Finally, P-A-18 /P-A-65/P-A-80 and P-A-83 were adapted with Anti-AFP McAb to establish a sensitivity assay for detection AFP by Sandwich ELISA. The limit of detection is 0.45 ng/mL and the linear rang is 1-100 ng/mL.VHH expression pHEN-VHH plasmids that were extracted from P-A-18/P-A-65/P-A-80 a