猪MHC Ⅱ类反式激活因子CIITA基因2个突变体的鉴定及其多克隆抗体的制备

发布时间：2018-03-31 21:11

本文选题：MHCⅡ类反式激活因子　切入点：基因突变体　出处：《广西大学》2012年硕士论文

【摘要】：猪瘟病毒(Classical swine fever virus, CSFV)是黄病毒科瘟病毒属的一个成员,所引起的猪瘟是一种高度传染性和致死性的疾病。近年的研究表明,猪瘟病毒在免疫猪的扁桃体、淋巴结和睾丸等多个器官持续性感染,猪瘟病毒在宿主体内的持续性感染是该病反复发作难以根绝的主要原因。 MHC Ⅱ类反式激活因子(Major Histocompatibility Class Ⅱ Transactivator, CIITA),是一个重要的共激活因子,其主要作用是通过蛋白的羧基端与结合到MHC Ⅱ类基因启动子上的多个转录因子相互作用,成为这些转录因子的支架,共同发挥对MHC Ⅱ类分子表达的调控作用。本实验室之前的研究结果表明,在猪瘟病毒感染的猪外周血淋巴细胞和体外培养的PK-15细胞中,主要组织相容性复合体Ⅱ (Major Histocompatibility Class Ⅱ, MHC Ⅱ)的表达水平均出现明显下调。CⅡTA是调节MHC Ⅱ表达的关键性分子,本研究的目的在于克隆猪CⅡTA基因,制备抗CⅡTA蛋白的多克隆抗体,使用制备的多克隆抗体检测真核转染的CⅡTA蛋白在PK-15细胞中的表达,探索CⅡTA蛋白的表达以及随后的MHC Ⅱ的表达与猪瘟病毒感染的关系,为进一步探索猪瘟病毒持续性感染的机制打下基础。本实验通过分离成年长白猪外周血淋巴细胞并抽提总RNA, RT-PCR扩增猪的CIITA基因,对扩增得到的猪CIITA基因的测序分析表明,我们获得了3条猪CIITA基因的序列,分别命名为CIITA4、CIITA8和CIITA-N,其中,CIITA4基因与参考基因(登录号为AY084053.1)的核苷酸序列的同源性达到99.4%,氨基酸同源性为98.6%；序列比对时我们还发现,CIITA8基因的核苷酸序列在其ORF的929-968位出现了40bp的碱基丢失的情况,CIITA-N基因的核苷酸序列在其ORF的225-368位和926-968位分别出现了144bp和40bp的碱基丢失；对CIITA8和CIITA-N进行氨基酸推导分析发现,因为926-968位基因片段的丢失,使得这两个基因的终止密码TGA提前,CIITA8和CIITA-N编码的蛋白质序列被缩短。所以本实验所获得的3条基因序列CIITA4、CIITA8和CIITA-N编码的蛋白质长度分别为565aa、312aa和264aa,初步推测这种核苷酸片段的丢失可能与CIITA基因的前体mRNA的选择性剪切有关。本研究扩增包含CIITA4基因3'端的723bp的核苷酸片段并将其亚克隆到原核表达载体PET-32a+上,成功构建PET-CIITA4-C原核表达载体并将其转化到大肠杆菌BL-21中进行IPTG的诱导表达。鉴定重组蛋白CIITA4-C的表达形式后,用Ni-NTA亲和层析的方法纯化重组蛋白并对其进行复性,复性后的重组蛋白免疫小鼠制备多克隆抗体并对制备的抗体进行反应性和效价的检测。同时,我们还构建了包含CIITA4基因ORF的真核表达载体PCDNA3.0-GFP-CIITA4,并成功在PK-15细胞上表达。使用制备的抗CIITA4蛋白的多克隆抗体检测转染了PCDNA3.0-GFP-CIITA4的PK-15细胞中的CIITA4蛋白的表达情况,并且探索了表达CⅡTA4蛋白后对猪瘟病毒在PK-15细胞中的复制的影响,结果表明,表达CⅡTA4蛋白后,猪瘟病毒E2蛋白的表达没有明显变化,CⅡTA4蛋白在PK-15细胞中的表达与猪瘟病毒感染的相互关系还需进一步研究。
[Abstract]:Classical swine fever virus (Classical swine fever virus, CSFV) is a member of the family Flaviviridae pestivirus, swine fever is caused by a highly contagious and lethal disease. Recent studies have shown that tonsil of classical swine fever virus in immunized pigs, lymph node and testis of multiple organ persistent infection of classical swine fever virus in the persistence of host infection is the main reason of recurrent disease difficult to eradicate.
MHC class II transactivator (Major Histocompatibility Class Transactivator II, CIITA), is an important coactivator, its main function is through the protein C-terminal and binding to MHC class II gene promoter on multiple transcription factors, these transcription factors become the support, to play the regulation effect on the expression of MHC class II molecules.
Our previous results showed that in PK-15 cells of classical swine fever virus infection of porcine peripheral blood lymphocytes and cultured in vitro, major histocompatibility complex (Major Histocompatibility, Class II, MHC II) expression level was significantly lower in.C II TA is a key molecule regulating the expression of MHC II, the research aim to clone porcine C TA gene, polyclonal antibody against C II TA protein expression, polyclonal antibody detection of eukaryotic transfection C II TA protein in PK-15 cells and explore the expression of C II TA protein and the expression of MHC II and the relationship between infection of classical swine fever virus and to further explore the mechanism of persistent HCV infection lay the foundation.
Through the experiments of peripheral blood lymphocytes from adult Landrace and extract the total RNA of porcine RT-PCR gene was amplified by CIITA, sequencing of porcine CIITA gene was amplified by the analysis showed that we obtained 3 sequences of porcine CIITA gene, named CIITA4, CIITA8 and CIITA-N, in the CIITA4 gene and reference gene (accession No. AY084053.1) nucleotide sequences homologous to 99.4% amino acid homology is 98.6%; we also found that the sequence alignment, the nucleotide sequence of the CIITA8 gene appeared 40bp base loss situation in 929-968 of its ORF, nucleotide sequence of the CIITA-N gene in the ORF of 225-368 and 926-968 respectively appeared 144bp and 40bp base were lost; the deduced amino acid analysis found on CIITA8 and CIITA-N, because the 926-968 gene fragments lost, this makes two gene stop codon of TGA, CIITA8 and CI The protein sequence of ITA-N encoding is shortened. So the obtained 3 gene sequences of CIITA4, CIITA8 and CIITA-N encoding the protein length were 565aa, 312aa and 264aa, speculated that the selective precursor of mRNA loss and CIITA gene the nucleotide fragment of the shear.
This study amplified nucleotide fragment containing the CIITA4 gene at 3'723bp and cloned into prokaryotic expression vector PET-32a+, prokaryotic expression vector PET-CIITA4-C and transformed into Escherichia coli expression induced by BL-21 in IPTG was successfully constructed. The identification of CIITA4-C recombinant protein expression and purification of recombinant protein and the renaturation by Ni-NTA affinity chromatography method, the detection system of mice immunized with recombinant protein complex after the preparation of polyclonal antibody and reactivity and titer of antibody preparation. At the same time, we also construct a eukaryotic expression vector PCDNA3.0-GFP-CIITA4 containing CIITA4 gene of ORF, and successfully expressed in PK-15 cells.
Detection of polyclonal antibody against CIITA4 protein was prepared using the PCDNA3.0-GFP-CIITA4 expression of CIITA4 protein in PK-15 cells, and to explore the expression of TA4 protein after C II effect of classical swine fever virus replication in PK-15 cells. The results showed that the expression of C II TA4 protein, no significant changes in expression of classical swine fever virus E2 protein, C II TA4 protein in PK-15 cells and the expression of classical swine fever virus infection relationship needs further study.

【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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