miR-122真核表达载体的构建、体外转录及其活性的鉴定

发布时间：2018-06-13 07:18

本文选题：miR-122 + 真核表达载体　；参考：《华中科技大学》2008年硕士论文

【摘要】： 目的 1.构建肝脏特异性表达的微小RNA——miR-122的真核表达载体; 2.将该重组质粒在人肝癌细胞系中转录,通过现代分子生物学技术鉴定其生物活性。 3.利用该重组质粒初步探讨miR-122与乙型肝炎病毒复制之间的关系。方法 1.真核表达载体的构建: 应用PCR技术,以从人类肝癌细胞中提取的基因组DNA为模板,获得miR-122的编码基因片段。根据设计的酶切位点,将其与真核表达载体pSuper同时双酶切,然后经T4连接酶连接,筛选得到稳定的克隆,命名为pHsa-m122。 2.鉴定重组体的生物活性: 以美国斯坦福大学医学院微生物与免疫教研室Peter Sarnow教授馈赠的荧光报告质粒GFP-122 sensor(GFP-122si)为报告质粒、GFP-124 sensor (GFP-124si)为无关对照质粒,该报告质粒GFP-122si的转录可被成熟的miR-122干扰,使绿色荧光蛋白的表达量下降,同样GFP-124si的转录也可被成熟的miR-124干扰,而不受成熟的miR-122干扰。因而将pHsa-m122与报告质粒GFP-122si共同转染人肝癌细胞系HepG2,以pHsa-m122与参照质粒GFP-124si共转染人肝癌细胞系HepG2为对照,分别用倒置荧光显微镜观察绿色荧光蛋白的表达、流式细胞仪检测细胞的平均荧光强度、Western blot检测绿色荧光蛋白的表达量这三种方式来检测GFP的表达变化,通过GFP蛋白表达量的下降来反映成熟的miR-122的表达情况。 3.初步探索miR-122与HBV复制之间的关系: 通过体外真核转染确定质粒pHsa-m122的有效性后,将pHsa-m122转染入人肝癌细胞系HepG2.2.15细胞,经过24小时后,用ELISA的方法来检测细胞分泌上清中HBsAg、HBeAg的表达量。结果 1.真核表达载体的构建: pHsa-m122经PCR鉴定,能够获得预期大小的目的片段; pHsa-m122经双酶切鉴定,可获得预期大小片段; pHsa-m122经Invitrogen公司测序证实,miR-122的编码基因片段已经正确插入pSuper载体中。 2.载体的生物活性鉴定: 荧光、流式、Western blot的结果均表明:pHsa-m122与报告质粒GFP-122si共同转染人肝癌细胞系HepG2,能够有效抑制绿色荧光蛋白的表达。因此证实pHsa-m122可转录出具有生物活性的成熟的miR-122; 3.载体的初步应用: 将pHsa-m122转染入人肝癌细胞系HepG2.2.15后,检测到细胞分泌上清中的HBsAg、HBeAg的表达量升高。结论 1. miR-122的前体序列成功地克隆到真核表达载体pSuper上,miR-122的真核表达载体pHsa-m122克隆成功; 2. pHsa-m122可以表达出具有生物活性的成熟的miR-122; 3.在对miR-122与HBV复制关系的初步探索中,我们的实验结果提示miR-122可能与乙型肝炎病毒的复制有一定关系。miR-122真核表达载体pHsa-m122的成功构建,为进一步研究miR-122在肝炎病毒复制及肝癌形成中的作用奠定了实验基础。本课题的创新点 1.在国内首次通过检测报告质粒表达绿色荧光蛋白的量的方法来检测微小RNA真核表达载体的生物活性,并运用荧光、流式、Western blot的方法来鉴定出微小RNA真核表达载体的生物活性。 2.在国内首次将微小RNA的真核表达载体运用到对乙型肝炎病毒复制的研究探索中。
[Abstract]:Purpose

1 . constructing a eukaryotic expression vector of a small RNA _ miR - 122 which is specifically expressed in the liver ;

2 . The recombinant plasmid is transcribed in human liver cancer cell line , and its biological activity is identified by modern molecular biology technique .

3 . The relationship between miR - 122 and replication of hepatitis B virus was investigated by using the recombinant plasmid .

method

1 . Construction of eukaryotic expression vector :

The gene fragment of miR - 122 was obtained from genomic DNA extracted from human liver cancer cells by using PCR technique . According to the designed enzyme cleavage site , it was digested with double enzyme at the same time as eukaryotic expression vector pSuper , then ligated with T4 ligase , screened to obtain stable clone , named pHsa - m122 .

2 . Identification of the biological activity of the recombinant :

The expression of green fluorescent protein was detected by an inverted fluorescence microscope , and the expression of green fluorescent protein was detected by Western blot . The expression of GFP - 122 was detected by Western blot .

3 . Initial exploration of the relationship between miR - 122 and HBV replication :

pHsa - m122 was transfected into HepG2.2 . 15 cell line HepG2.2 . 15 . After 24 hours , the expression of HBsAg and HBeAg was detected by ELISA .

Results

1 . Construction of eukaryotic expression vector :

pHsa - m122 was identified by PCR to obtain the desired fragment of the desired size ; pHsa - m122 was identified by double enzyme digestion to obtain the desired size fragment ; pHsa - m122 was confirmed by sequencing by Invitrogen Corporation , and the encoded gene fragment of miR - 122 was correctly inserted into the pSuper vector .

2 . Identification of the biological activity of the carrier :

The results of fluorescence , flow and Western blot showed that pHsa - m122 co - transfected human liver cancer cell line HepG2 with reporter plasmid GFP - 122 si , which can effectively inhibit the expression of green fluorescent protein . Therefore , it was confirmed that pHsa - m122 could transcribe mature miR - 122 ; 3 . vector preliminary application :

After transfection of pHsa - m122 into human hepatoma cell line HepG2.2 . 15 , the expression of HBsAg and HBeAg in the supernatant was detected .

Conclusion

1 . The precursor sequence of miR - 122 is successfully cloned into the eukaryotic expression vector pSuper , and the eukaryotic expression vector pHsa - m122 of miR - 122 is cloned successfully ;

2 . pHsa - m122 can express the mature miR - 122 with biological activity ;

3 . In the preliminary exploration of the relationship between miR - 122 and HBV replication , our experimental results suggest that miR - 122 may be associated with replication of hepatitis B virus . The successful construction of miR - 122 eukaryotic expression vector pHsa - m122 provides an experimental basis for further research on the role of miR - 122 in the replication of hepatitis virus and the formation of liver cancer .

Innovation Points of the Project

1 . The biological activity of the eukaryotic expression vector was first detected by detecting the amount of green fluorescent protein expressed in the reporter plasmid at home for the first time , and the bioactivity of the eukaryotic expression vector was identified by fluorescence , flow and Western blot .

2 . For the first time , the eukaryotic expression vector of microRNA was applied to the study of hepatitis B virus replication .
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R346

【参考文献】