【作者】 李强；

【导师】 李学伟；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 天然免疫是机体防御微生物病原体入侵第一道防线,机体细胞通过表达不同的模式识别受体（Pattern recognition receptors,PRRs）,来识别包括类脂,脂蛋白,蛋白和核酸在内的各种病原相关分子模式（Pathogen-associated molecular patterns,PAMPs）,激活机体细胞内信号通路,诱导炎症细胞因子和趋化因子及干扰素的表达,上调细胞表面MHCⅡ类分子和共刺激分子从而参与适应性免疫应答。Toll样受体（Toll-like receptors,TLRs）是天然免疫系统中重要的病原模式识别受体之一,也是典型的Ⅰ类跨膜受体,至今为止,已经在小鼠上发现了13个TLRs家族成员,人上11个。TLRs可以识别包括细菌,病毒,原生动物和真菌在内的多种微生物体的病原体相关分子模式,在机体天然免疫中发挥着重要的作用。Toll样受体7（Toll-like receptor 7,TLR7）是TLRs家族的重要成员之一,可以通过识别病毒的单链RNA（ssRNA）诱导机体表达干扰素α（IFN-α）和白细胞介素12（IL-12）等细胞因子,对病毒做出相应的免疫应答。现已证实TLR7可介导多种病毒的免疫应答,包括HIV-1,流感病毒,VSV（泡状口腔炎病毒）和HCV（丙型肝炎病毒）等。髓样分化因子88（Myeloid differentiation primary response protein 88,MyD88）是TLRs和IL-1Rs信号传导途径中重要的接头蛋白。近来研究证实MyD88还参与了IFN-γ诱导细胞响应,并且对IFN-γ诱导的mRNA的稳定性有着重要的作用。本研究应用RT-PCR、简并PCR、RACE、巢式PCR技术从猪肠系膜淋巴结组织总RNA中克隆出猪TLR7的cDNA序列。分析结果表明,克隆到的序列全长3 834 bp（GenBank收录号:EF469730）,其3 153bp的开放阅读框编码1 050个氨基酸残基的猪Toll样受体7蛋白。推导的氨基酸序列分析显示,在猪TLR7的胞外区,具有多LRR-RI结构域,胞内具有TIR结构域,表现出典型的TLRs家族结构特征,同源性分析结果显示,TLR7在进化过程中具有高度保守性,与牛、狗、马、人、猫和小鼠的氨基酸序列同源性分别为:90.8%、87.4%、87.2%、84.9%、86.7%和78.2%。采用Real time PCR技术对太湖猪TLR7基因在心、脑、肺、肝、脾、肾、骨骼肌、脂肪、肠系膜淋巴结、空肠和派伊氏结11个组织中的表达谱进行了检测分析。结果显示,猪TLR7主要表达于肠系膜淋巴结和派伊氏结等组织。在皮下脂肪、心和骨骼肌组织中表达量相对较少。应用RT-PCR技术从猪肠系膜淋巴结组织总RNA中克隆出猪MyD88的cDNA序列,我们从三个不同成年太湖母猪的肠系膜淋巴结组织克隆该序列来验证这个序列。序列分析显示克隆到的猪MyD88 cDNA序列长897bp,包含猪MyD88基因全CDS区。该核酸序列已提交到GenBank（GenBank收录号:EF198416）。猪MyD88基因开放式阅读框编码293个氨基酸残基的蛋白质序列。我们利用在线软件SMART程序对推导的氨基酸序列分析其功能结构域,结果表明,该蛋白具有典型的MyD88结构,N端具有死亡结构域,中间的连接结构域和C末端的TIR结构域。同源分析表明,猪MyD88蛋白氨基酸序列也具有高度的保守性,与人、牛、小鼠和大鼠的同源性分别为88.4%、85.7%、78.8%和78.2%。猪MyD88与牛和人的遗传距离较近,与小鼠相对较远,进化树表明猪先与人和牛聚为一类,然后再与小鼠和大鼠聚类,遗传距离分析也显示了相似的结果。利用Real time PCR技术对太湖猪11个组织的表达谱分析显示猪MyD88基因广泛表达于各组织中,在派伊氏结、肠系膜淋巴结组织中表达量较大,在心脏和骨骼肌组织中表达量相对较少。Pichia pastoris是目前应用最广泛的外源蛋白表达系统之一。该表达系统兼有原核表达系统和真核表达系统的优点:①与酿酒酵母相似,操作简单;②不论胞内表达还是分泌表达,表达量高;③能对外源真核基因进行正确翻译和翻译后加工与修饰,包括蛋白质的糖基化,二硫键的形成以及蛋白的水解;④表达试剂盒商业化。本实验我们选用pPIC9K作为表达载体,KM71为宿主菌,在猪MyD88序列两端分别加上EcoRⅠ和NotⅠ两个酶切位点,终止密码子前加上六个组氨酸标签,将序列克隆到T载体,提质粒双酶切后利用T4 DNA连接酶克隆到pPIC9K上,利用SacⅠ酶切成单链后电转化导入酵母KM71中,利用组氨酸缺陷型MD平板和含不同浓度G418的YPD平板进行筛选,得到了不同抗G418水平的阳性转化子,将不同抗性的阳性转化子用甲醇诱导表达,首次成功利用毕赤酵母表达体系表达出猪MyD88蛋白,并应用Bio-Rad蛋白质纯化仪,GE的HisTrap^TM HP对含有表达蛋白的上清液利用亲和层析进行纯化,SDS-PAGE电泳显示在分子量为34KDa附近有一条明显的电泳带,与生物信息学分析的猪MyD88表达蛋白分子量（包括His6标签）34..06KDa结果一致。更多还原

【Abstract】 The innate immune system is the first line of the defensive mechanisms that protect hosts from invading microbial pathogens.Host cells express various pattern recognition receptors（PRRs） that sense diverse pathogen-associated molecular patterns（PAMPs）,ranging from lipids, lipoproteins,proteins and nucleic acids.Recognition of PAMPs by PRRs activates intracellular signaling pathways that culminate in the induction of inflammatory cytokines,chemokines, interferons（IFNs） and upregulation of MHC-Ⅱand co-stimulatory molecules to influence the adaptive immune system.Toll-like receptors（TLRs） are one of the important PRRs families and are typeⅠmembrane proteins.To date,13 mouse TLRs and 11 human TLRs have been identified, and each TLR appears to recognize distinct PAMPs derived from various microorganisms, including bacteria,viruses,protozoa and fungi.As a member of Toll-like receptor（TLR） family,Toll-like receptor 7（TLR7） can identify the single strand RNA of virus and help the host to take corresponding control of immune response by inducing to some cytokines expression in animal body such as interferon（IFN）-αand interleukin（IL）-12.It was demonstrated that some viruses（Including vesicular stomatitis virus, influenza virus,Human immunodeficiency virus-Ⅰand Hepatitis C Virus etc） stimulate immune responses through TLR7.Myeloid differentiation primary response protein 88（MyD88） is an important adapter protein in the signal transduction pathway mediated by interleukin-1（IL-1） and Toll-like receptors.New evidence shows that MyD88 also participates in interferon-γ-induced cellular responses and has a role to prolong the half-time of some mRNA induced by interferon-γ.Here we cloned cDNA of TLR7 gene from total RNA of mesenteric lymph nodes（MLN） in Neijiang pig by RT-PCR,and RACE（rapid amplification of cDNA ends）.Sequence analysis indicated that the porcine TLR7 cDNA cloned was 3834 nt in length（GenBank accession number EF469730） and the open reading frame encodes a deduced protein with 1050 amino acids residues. The analysis of deduced amino acids sequence indicated that TLR7 is a typical typeⅠtransmembrane protein with multi-LRR-RI ectodomains and a TIR cytoplasmic domains,the common structural features of TLRs.The comparison of the deduced amino acids sequence of porcine TLR7 with those of cattle,dog,human,cat and mouse showed that the amino acids homology were 90.8%,87.4%,84.9%,86.7%and 78.2%respectively and the TLR7 is highly conserved among the different mammal species.Using real-time quantitative PCR,we detected TLR7 mRNA expression in a panel of porcine tissues（heart,lung,spleen,liver,kidney,skeletal muscle,brain,jejunum,Peyer’s patches and mesenteric lymph nodes） with the greatest levels of expression observed in mesenterie lymph nodes（MLN）,and Peyer’s patches.Pocine MyD88 cDNA was cloned using mRNA isolated from adult swine mesenteric lymph nodes（MLN）.The sequences were confirmed in three mesenteric lymph nodes from various adult.Nucleotide sequencing of pocine MyD88 revealed a 897bp cDNA sequence.Including the pocine MyD88 structural gene.The nucleotide sequence of pocine MyD88 has been submitted to the GenBank nucleotide databases（GenBank accession number EF198416）.A predicted open reading flame（ORF） lay between bp 45 and 926 and encoded a 293aa protein.To determine the structural domains in pocine MyD88.the amino acid sequence was analyzed using the SMART program.The deduced amino acid sequence of pocine MyD88 possesses a typical MyD88 domain including an N-terminal death domain.as well as interm ediate and C-terminal TIR domains.Two other regions were intrinsically disordered.The alignment of amino acid sequences for MyD88 showed that the pocine MyD88 sequence is highly conserved among sequences from other species.The amino acid sequence of the pocine MyD88 is 88.4%、85.7%、78.8%and 78.2% identical to that of human,cattle rat and of mouse MyD88,respectively.These results indicated that pocine MyD88 is more similar to cattle and human than to mouse MyD88 at aminoacid levels.Phylogenetic analysis showed that pocine MyD88 belonged to the group containing cattle MyD88 and human MyD88.In addition,pocine MyD88 was more closely related to human MyD88 and cattle MyD88 than to mouse MyD88 in terms of identity.Real-time PCR analysis shows that the MyD88 gene is expressed in various tissues,but at different levels.The expression levels of this gene are higher in Peyer’s patches and mesenteric lymph nodes（MLN）.The expression levels in musle and heart are very lower and aren’t almost be detected.Pichia pastoris is one of the most used expression systems for the high level expression of heterologous proteins.This expression system has the advantages of Prokaryotic systems expression and Eukaryotic expression systems.①the simplicity of techniques needed for the molecular genetic manipulation of P.pastoris and their similarity to those of Saccharomyces cerevisiae,one of the most well-characterized experimental systems in modern biology;②the ability of P.pastoris to produce foreign proteins at high levels,either intracellularly or extracellularly;③the capability of performing many eukaryotic posttranslational modifications, such as glycosylation,disulfide bond formation and proteolytic processing;and④the availability of the expression system as a commercially available kit.In our studies,we used pPIC9K as expression vector and KM71 as host strains to express porcine MyD88 protein.After the porcine MyD88 gene with EcoRⅠand NotⅠrestriction Sites and His tag was cloned to pMD18-T vector, pMD18-T-MyD88 and pPIC9K were cutted by EcoRⅠand NotⅠand the cutted fragments were ligated by T4 DNA ligase.The porcine MyD88 gene expression vector,pPIC9K-MyD88,was constructed successfully after sequencing.The expression vector pPIC9K-MyD88 used for transformation,was linearized by SacⅠ.Pichia pastoris KM71 strains were made competent and transformed with SalⅠ-linearized pP9K-MyD88 by electroporation.After selection by MD plates, YPD plates containing G418,some positive colonies which exhibited different anti-G418-levels were obtained.We selected different anti-G418-levels positive clones and induced them to expression porcine MyD88 protein by methanol.The culture supernatant contained the target protein was purifed by affinity chromatagraphy used HisTrap^TM HP and BioLogic DuoFlow. Purified MyD88 expression products were analyzed by SDS-PAGE and found a major protein band at a molecular weight of 34 kDa,which is consistent with the molecular weight of pig MyD88.The porcine MyD88 was expressed successfully.更多还原