【作者】 杨明夏；

【导师】 朱昌亮；

【作者基本信息】 南京医科大学 ， 病原生物学， 2003， 硕士

【摘要】 媒介昆虫对杀虫剂的抗性是虫媒病控制中一个亟待解决的突出问题。研究表明，抗性产生是由基因决定的。本实验室运用SSH结合cDNA芯片鉴定出NYD-Ch和NYD-Tr基因在淡色库蚊溴氰菊酯抗性品系中差异性高表达。 本研究根据本实验室获得的全长NYD-Ch和NYD-Tr基因序列，运用PCR方法从构建好的抗性品系cDNA文库中扩增出两基因完整开读框，通过基因重组方法，成功构建NYD-Ch和NYD-Tr基因原核和真核表达载体，并初步探讨了两基因的原核诱导表达。为后续研究NYD-Ch和NYD-Tr基因抗性品系中高表达在淡色库蚊抗性产生中的分子机制、媒介抗性产生机理以及媒介综合防治奠定基础。 第一部分：抗性相关基因NYD-Ch和NYD-Tr原核、真核表达载体的构建 根据本实验室报道的在淡色库蚊溴氰菊酯抗性品系中高表达的NYD-Ch和NYD-Tr基因的cDNA全长序列，设计引物，从本实验室构建的淡色库蚊溴氰菊酯抗性品系全长cDNA文库中，通过PCR方法获取基因的完整开读框序列。引物设计时，根据原核表达载体pET-28a(+)和真核表达载体pcDNA3.1(+)、pEGFP-C3表达的需要，在基因起始密码子ATG前加入酶切位点的同时，注意补加碱基，以确保不发生读码框移。PCR产物克隆入pGEM-Teasy载体，通过PCR、酶切和基因测序鉴定。 小量抽提pGEM-T／NYD-Ch和pGEM-T／NYD-Tr重组质粒，双酶切切下目的基因片段NYD-Ch和NYD-Tr。小量抽提pET-28a(+)和pcDNA3.1(+)、pEGFP-C3质粒，双酶切后，按照一定比例在T4DNA链接酶作用下，与基因片段hYD-Ch和NYD-Tr进行基因重组。通过PCR、双酶切方法鉴定重组表达质粒，基因测序验证。 结果：pGEM-T／NYD-Ch和pGEM-T／NYD-Tr基因测序表明，所 雨京王科人学帅卜学应沦义扩增基因为所需要的目的NYD－Ch和NYD－Tr基因，引物设计酶切位点正确。重纽表达质粒PEl28a（＋）／：NyJJ－Ch、PET28a（＋）／NYD－Tr；pcDNA3．l（＋）fN－YD－Ch、pcDNA3月什＊NYD＊r和 pEGFP－C3MD－Ch、pEGFP－C3／NYD丁r，用基因特异性弓z物和载体引物PCR扩增，都能得到目的片段；双酶切能切下目的基因片段。结果表明，重组表达质粒中目的基因片段未发生框移，与融合基固同框表达。第二部分：多克隆抗＊＊Y和＊RY抗体的制备和抗性的初步检测 用牛＊HY和＊＊Y蛋白，通过长程免疫小鼠白 方二，制备小鼠抗牛＊＊Y和＊RY多克隆抗体。等比伊卜昆合弗氏完全佐剂和牛＊＊Y及T班成油包水状，小鼠背部皮下多点注射，15天后用弗氏不完全佐剂与抗原等比例 i昆合加强免疫，再 10天后小鼠尾部采血，用 ELISA方法检测小鼠抗牛Cw和T队抗体哈G的滴度。再5天后完全抗原加强免疫，整个免疫过程共45天后眼球取血分离小鼠抗血清，ELISA检测抗体滴度，血清标记分装，以备后续实验以及原核表达鉴定用，并以此多抗 Western blot检测抗性和敏感品系淡色库蚊成虫蛋白，探讨进行抗性检测的可能性。 结果：用 ELISA方法检坝IJ抗体滴度。纯抗原按每孔 15 P g包板，抗血清从1习OO开始倍比稀释，显色反应后酶标仪检测吸光度，结果显示；，J、鼠抗牛 Cw和 T盯的多克隆抗体滴度都能达到 1：10000。成蚊粗提蛋白Western blot显色反应后，观察到抗TRY组抗性品系出现一条大约28m显色条带，与NYD－Tr基因翻译蛋白大，J件 当。但是在抗CHY组没有观察fu。提示采用CHY和TRY抗体检测蚊虫抗性是可行的，值得进一步研究。第三部分：抗药性相关基因NY D－Ch和N－YD－T：原核诱导表达和鉴定 从经过鉴定的阳性克隆菌JM mg 中抽提重组表达质粒pET28a什）／NYD－Ch、pET28a幻／NYD＊卜 用冰冷CaCI，法转入表达茵种 BLZI（DE3）中，经 ImMrTG诱导后，收吴细菌，力。100… 南京医科人学硕上学位论义lx Loading Buffer 00。C煮沸变性后，SDS－PAGE蛋白凝胶电泳，考马斯亮兰染色观察表达蛋白大小。阳性样本重新SDS－PAGE胶电泳后，电转膜至＊C膜上后，用第二部分制备的小鼠抗血清作为一抗，辣根过氧化物酶标羊抗小鼠 IgG抗体作为＝抗，通过 Western blot方法初步鉴定蛋白诱导表达。 结果：用I二M的IPTG诱导菌液2小时后，与空载相比能看到一个大约 33kD的蛋白条带，和设计一致。目的 NYD－Ch和NYD＊r基因推导蛋白质分子大小分别为29to和28．4 kDkD；加上载体融合部分，总蛋白分子量约 33kD左右。Western blot检测显示。在 NC膜上分子量约33m处有明显的显色条带，与SDS－PAGE蛋白电泳胶上蛋白条带一致，初步鉴定表达蛋白为目的蛋白。实验结果为后续研究基因NYD－Ch和NYD－TY高表达在抗性产生中的分子机命卜及进一步的抗性研究奠定了基础。更多还原

【Abstract】 The resistance to insecticide played a serious role in the control of pests. All the research gives more evidence that the resistance to insecticide in pest is determined by gene. NYD-Ch gene and NYD-Tr gene were isolated and identified from the mosquito, Cx. pipiens pallens, by suppression subtractive hybridization (SSH) combined with cDNA chip, which were highly expressed in the deltamethrin-resistant strain compared in the susceptible strain. And this study is to construct the prokaryotic and eukaryotic expression plasmids of gene NYD-Ch and NYD-Tr, to obtain the polyclonal antibody of mouse anti-bovine CHY and TRY, and to learn prokaryotic expressions of the two genes.Part one: Construction and identification of expression plasmids of NYD-Ch gene and NYD-Tr geneThe complete open reading frames (ORFs) of NYD-Ch gene and NYD-Tr gene were amplified from the full-length cDNA express library of the deltamethrin-resistant strain of Cx.pipiens pallens by PCR according to the reported sequences of our laboratory. The enzyme restriction sites were added at the head of ATG of the two genes considering the express of the prokaryotic plasmid pET-28a(+) and the eukaryotic plasmids pcDNA3.1(+) and pEGFP-C3 when designing the primers of the two genes. The PCR fragments were cloned into pGEM-T easy vector, and then identified by PCR, enzyme cutting and gene sequencing. Cut from the re-construction plasmid pGEM-T/NYD-Ch andpGEM-T/NYD-Tr, and from the super coil plasmids pET-28a(+), pcDNA3.1(+) and pEGFP-C3, the fragments NYD-Ch , NYD-Tr, pET-28a(+), pcDNA3.1(+) and pEGFP-C3 were obtained with the enzyme restriction site ends. The gene and plasmid fragments were re-combined in the condition of T4 DNA ligase on the basis of some ratio. And then the re-construction plasmids were identified using PCR, double enzyme cutting and gene sequencing. The results showed that NYD-Ch gene and NYD-Tr gene sequences were the same as the reported, indicating that the express plasmids pET-28a(+)/NYD-Ch,pET-28a(+)/NYD-Tr, pcDNA3.1(+)/NYD-Ch, pcDNA3.1(+)/NYD-Tr, pEGFP-C3/NYD-Ch and pEGFP-C3/NYD-Tr were re-constructed successfully.Part two: Preparation of polyclonal antibody of mouse anti-CFTY and anti-TRY, and preliminary detection of resistance in mosquitoThe bovine CFFY and TRY mixed equally with the Freund’s complete adjuvant were used to immunize the mice separately. 15 days later, the antigen mixed equally with the Freund’s incomplete adjuvant was injected under the dermis of mice on the back to enhance the effect of immune. And then 10 days later, the serum of antibody titer was determined primarily using ELISA, and 5 days latter again, the complete antigen alone was used to immunize the mice. After 45 days from the first immune, the serum of mice was obtained and the titer of antibody was determined by ELISA. During the course of the immune, the dose of antigen was equal. The antibody was used to study the resistance in the mosquito elementarily by Western blot. The results show that high titer of the polyclonal antibody of mouse anti-CHY and anti-TRY were prepared and the titer was more than 1:10000. And a tape of protein about 28kD was observed on the NC membrane of resistance strain when the firstantibody was anti-TRY polyclonal antibody. The protein was coincident with the protein of NYD-Tr. But in the group of anti-CHY, none was appeared in the resistant strain.Part three: Prokaryotic expression and identification of resistance associated NYD-Ch and NYD-Tr geneThe plasmids of pET-28a(+)/NYD-Ch and pET-28a(+)/NYD-Tr were transfected into the E.coil BL21(DE3) by CaCb, and then induced by ImM IPTG to obtain the protein in different time. Mixed with the 1 X Loading Buffer and degenerated in the condition of 100癈 water , the sediment of bacterium were separated by SDS-PAGE and then dyed with Coomassie Light blue to learn the molecular weight of the express protein. And then the positive sample was identified by the Western blot using the polyclonal antibody of mouse anti-CHY and anti-TRY a更多还原