【作者】 朱晔斌；

【导师】 张文成；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 目的:克隆和分析ZFP580基因cDNA开读框架全编码区(编码172个氨基酸)、N端编码区(编码1-88位氨基酸)、C端C2H2型锌指主构域编码区(编码89-172位氨基酸)。构建ZFP580基因原核表达载体并鉴定,诱导表达GST-ZFP580融合蛋白。方法:1从正常大鼠小脑组织中提取总RNA。2根据GenBank公布的大鼠ZFP580基因cDNA开读框架序列设计引物,用RT-PCR的方法扩增ZFP580基因cDNA开读框架以及ZFP580基因的N端编码区和C端编码区。3用T-A克隆方法将目的基因克隆至pMD18-T-Easy载体中,挑取琼脂板上的白色菌落,接种于5ml含有Amp的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备重组质粒。采用BglII和XhoI双酶切法、PCR法及测序法鉴定重组质粒。构建成功的阳性重组质粒记作pMD18-T-ZFP580。同法成功克隆ZFP580基因N端编码区和C端编码区,阳性重组质粒记作pMD18-T-N、pMD18-T-C。4用BglII和XhoI对克隆载体pMD18-T-ZFP580和原核表达载体pET-42a(+)分别进行双酶切,琼脂糖凝胶电泳酶切产物,纯化回收带有粘端的ZFP580基因和pET-42a(+)载体。将ZFP580基因和pET-42a(+)表达载体用T4DNA连接酶连接过夜。连接产物转化DH5α感受态细胞,加LB液体培养基,37℃培养60min。将菌液均匀涂布于含Kan的LB琼脂平板上,于37℃培养箱中培养12-16h。挑取琼脂板上的单菌落,接种于5ml含有Kan的LB液体培养基中,37℃摇菌培养过夜。用碱裂解法小剂量制备质粒进行鉴定。阳性重组质粒记作pET-42a-ZFP580。同法成功构建ZFP580基因的C端编码区,阳性重组质粒记作pET-42a-C。阳性重组质粒用双酶切的方法鉴定并双向测序,确认有无读码框错误。5阳性重组载体pET-42a-ZFP580转化E.coli BL21(DE3),IPTG诱导表达,优化表达条件,确定最佳表达参数,SDS-PAGE分析鉴定GST-ZFP580蛋白的表达。6生物信息学分析ZFP580基因结构和功能。结果:1甲醛变性琼脂糖凝胶电泳呈现清晰的28S和18S两条带,并且28S宽度以及亮度是18S的2倍,5S量较少。证实所提取总RNA完整性高。测OD260/OD280比值1.9左右,证明总RNA纯度高,无蛋白质和酶等抑制物残留。2 RT-PCR扩增后,在519bp附近出现了一条特异性条带,此DNA条带与大鼠ZFP580基因cDNA开读框架全编码区的大小相符合。测序证明已获得ZFP580基因开读框架,其序列与GenBank中预测序列完全一致。同法获得264bp和252bp大鼠ZFP580基因的N端编码区和C端编码区。序列比对结果证实与GenBank中预测的一致。3 T-A克隆构建获得的阳性克隆载体pMD18-T-N、pMD18-T-C、pMD18-T-ZFP580,用Bgl II和Xho I双酶切和PCR法鉴定。结果表明,上述三个克隆载体已成功构建。4原核表达载体pET-42a-ZFP580、pET-42a-C经鉴定证实构建成功。测序证实pET-42a-ZFP580载体无读码框的错误,转化入表达菌株E.coli BL21(DE3), IPTG诱导,SDS-PAGE鉴定分析GST-ZFP580融合蛋白表达情况。5小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。深入探讨了未能表达目的蛋白的原因。6大鼠ZFP580基因cDNA全长1100bp,完整开读框架为519bp,编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域:N端的富含脯氨酸结构域和C端的3个C2H2型锌指结构。结论:1大鼠ZFP580基因cDNA开读框架全编码区、大鼠ZFP580基因的N端编码区和C端编码区与GenBank中预测序列完全一致。2成功构建克隆载体pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580和原核表达载体pET-42a-ZFP580、pET-42a-C。3原核表达载体pET-42a-ZFP580读码框正确无误,IPTG诱导表达,经SDS-PAGE分析表达情况。小剂量多次进行蛋白表达实验,未能诱导表达目的蛋白。4详细分析大鼠ZFP580基因结构和功能,大鼠ZFP580基因开读框架编码一个含有172个氨基酸的蛋白质,所编码的蛋白与锌指蛋白相似有2个功能结构域。更多还原

【Abstract】 Objective: To clone the full-length open reading frame cDNA sequence of Zinc Finger Protein (ZFP580, encoding 172 amino acids) gene from rat cerebellum. To clone and analyze the cDNA sequence of amino terminal encoding region (encoding 1-88 amino acids) and carboxyl terminal encoding region (encoding 89-172 amino acids) of ZFP580 gene. To construct prokaryotic fusion expression vectors and induces the expression of fusion protein GST-ZFP580.Methods: 1 Total RNA was extracted from rat cerebellum with Trizol solution.2 The specific primers were designed according to the cDNA encoding sequence of ZFP580 full-length open reading frame published by GenBank. The cDNA encoding sequences of ZFP580 full-length open reading frame, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR. Sequence analysis of these fragments and its deduced amino acids were accomplished online at the National Center for Biotechnology Information servers and edited using the Primer5,Clustalx and DNAMAN programs.3 These cDNA fragments of ZFP580 gene were cloned into pMD18-T-Easy vector according to the instructions of the T-A clone kit and sequenced. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Xho I and Bgl II to select positive recombinants. The recombinant plasmids were named as pMD18-T-ZFP580, pMD18-T-C and pMD18-T-N respectively. The recombinant plasmids were sent to Takara Company to be sequenced by using the dideoxy chain-termination method.4 The pMD18-T-ZFP580 recombinant vector and pET-42a(+) fusion expression vector was digested by the Bgl II and Xho I respectively. The digested products were purified, recoverd and then linked by T4DNA ligase at 16°C for 1h. The recombinants were then transformed into E.coli DH5αcompetent cells. Several clones were randomly selected, then inoculated in 5mL LB liquid culture medium(containing Kan, 10μg/ml) respectively, and cultured 37°C at 220 rpm overnight. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Bgl II and Xho I to select positive recombinants. The cDNA encoding sequence of ZFP580 full-length open reading frame was sub-cloned into prokaryotic expressing plasmid pET-42a (+), generating pET-42a-ZFP580. Prokaryotic expression vector pET-42a-ZFP580 was sequenced by using the dideoxy chain-termination method and confirmed by reading both strands.5 Prokaryotic expression recombinant plasmids were transformed into E.coli BL21(DE3) to express the proteins. After the recombinant bacterium was induced with IPTG, the expressed recombinant protein was analyzed with SDS-PAGE.Results: 1 The total RNA product was examined by the electrophoresis of agarose gel containing formaldehyde. Results showed that there were two clear bands of 18S and 28S.The ratio A260/A280 and A260/A230 of RNA sample was all about 1.9, which demonstrated that RNA quantity and purity were well.It can be used in the following RT-PCR.2 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR successfully. DNA sequencing results showed that the ZFP580 open reading frame sequence, the sequences of ZFP580 amino terminal encoding region and ZFP580 carboxyl terminal encoding region were exactly consistent with the sequence reported in GenBank.3 The results of double enzyme digestion with Bgl II and Xho I showed the recombinant plasmids construction of pMD18-T- ZFP580, pMD18-T-C and pMD18-T-N of ZFP580 gene is successful.4 The recombinant plasmids of pET-42a-ZFP580 and pET-42a- C were confirmed by restriction endonuclease digestion and DNA sequencing respectively. The result indicates that the recombinant plasmid of pET-42a-ZFP580 contains the correct open reading frame.5 The expression of the GST-ZFP580 fusion protein in E.coli BL21 (DE3) was analyzed by SDS-PAGE. No expected protein band was observed with induction of IPTG6 The sequence of the whole length cDNA of ZFP580 gene is 1100bp. It contains an open reading frame (519bp), encoding a protein consisted of 172 amino acids. The result of amino acids sequence analysis of zinc finger protein online demonstrated that the encoding protein of ZFP580 has two domains. The amino terminal region between amino acids 5 and 88 is also remarkable rich in Proline residues. The carboxyl terminal region between amino acids 94-172 includes three high conserved C2H2 Zinc finger motifs.Conclusion: 1 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were successfully cloned. Those cDNA sequences are identical to the predicted ZFP580 gene of GenBank.2 The clone vectors of pMD18-T-N、pMD18-T-C、pMD18-T- ZFP580 and pET-42a-ZFP580 fusion expression vector were constructed successfully.3 No expected protein band was observed with induction of IPTG.4 The open reading frame of ZFP580 gene encodes a 172 amino acid protein containing two domains.更多还原