本文選題：MHCⅡ類反式激活因子　切入點(diǎn)：基因突變體　出處：《廣西大學(xué)》2012年碩士論文

【摘要】：豬瘟病毒(Classical swine fever virus, CSFV)是黃病毒科瘟病毒屬的一個(gè)成員,所引起的豬瘟是一種高度傳染性和致死性的疾病。近年的研究表明,豬瘟病毒在免疫豬的扁桃體、淋巴結(jié)和睪丸等多個(gè)器官持續(xù)性感染,豬瘟病毒在宿主體內(nèi)的持續(xù)性感染是該病反復(fù)發(fā)作難以根絕的主要原因。 MHC Ⅱ類反式激活因子(Major Histocompatibility Class Ⅱ Transactivator, CIITA),是一個(gè)重要的共激活因子,其主要作用是通過蛋白的羧基端與結(jié)合到MHC Ⅱ類基因啟動(dòng)子上的多個(gè)轉(zhuǎn)錄因子相互作用,成為這些轉(zhuǎn)錄因子的支架,共同發(fā)揮對(duì)MHC Ⅱ類分子表達(dá)的調(diào)控作用。 本實(shí)驗(yàn)室之前的研究結(jié)果表明,在豬瘟病毒感染的豬外周血淋巴細(xì)胞和體外培養(yǎng)的PK-15細(xì)胞中,主要組織相容性復(fù)合體Ⅱ (Major Histocompatibility Class Ⅱ, MHC Ⅱ)的表達(dá)水平均出現(xiàn)明顯下調(diào)。CⅡTA是調(diào)節(jié)MHC Ⅱ表達(dá)的關(guān)鍵性分子,本研究的目的在于克隆豬CⅡTA基因,制備抗CⅡTA蛋白的多克隆抗體,使用制備的多克隆抗體檢測(cè)真核轉(zhuǎn)染的CⅡTA蛋白在PK-15細(xì)胞中的表達(dá),探索CⅡTA蛋白的表達(dá)以及隨后的MHC Ⅱ的表達(dá)與豬瘟病毒感染的關(guān)系,為進(jìn)一步探索豬瘟病毒持續(xù)性感染的機(jī)制打下基礎(chǔ)。 本實(shí)驗(yàn)通過分離成年長白豬外周血淋巴細(xì)胞并抽提總RNA, RT-PCR擴(kuò)增豬的CIITA基因,對(duì)擴(kuò)增得到的豬CIITA基因的測(cè)序分析表明,我們獲得了3條豬CIITA基因的序列,分別命名為CIITA4、CIITA8和CIITA-N,其中,CIITA4基因與參考基因(登錄號(hào)為AY084053.1)的核苷酸序列的同源性達(dá)到99.4%,氨基酸同源性為98.6%；序列比對(duì)時(shí)我們還發(fā)現(xiàn),CIITA8基因的核苷酸序列在其ORF的929-968位出現(xiàn)了40bp的堿基丟失的情況,CIITA-N基因的核苷酸序列在其ORF的225-368位和926-968位分別出現(xiàn)了144bp和40bp的堿基丟失；對(duì)CIITA8和CIITA-N進(jìn)行氨基酸推導(dǎo)分析發(fā)現(xiàn),因?yàn)?26-968位基因片段的丟失,使得這兩個(gè)基因的終止密碼TGA提前,CIITA8和CIITA-N編碼的蛋白質(zhì)序列被縮短。所以本實(shí)驗(yàn)所獲得的3條基因序列CIITA4、CIITA8和CIITA-N編碼的蛋白質(zhì)長度分別為565aa、312aa和264aa,初步推測(cè)這種核苷酸片段的丟失可能與CIITA基因的前體mRNA的選擇性剪切有關(guān)。 本研究擴(kuò)增包含CIITA4基因3'端的723bp的核苷酸片段并將其亞克隆到原核表達(dá)載體PET-32a+上,成功構(gòu)建PET-CIITA4-C原核表達(dá)載體并將其轉(zhuǎn)化到大腸桿菌BL-21中進(jìn)行IPTG的誘導(dǎo)表達(dá)。鑒定重組蛋白CIITA4-C的表達(dá)形式后,用Ni-NTA親和層析的方法純化重組蛋白并對(duì)其進(jìn)行復(fù)性,復(fù)性后的重組蛋白免疫小鼠制備多克隆抗體并對(duì)制備的抗體進(jìn)行反應(yīng)性和效價(jià)的檢測(cè)。同時(shí),我們還構(gòu)建了包含CIITA4基因ORF的真核表達(dá)載體PCDNA3.0-GFP-CIITA4,并成功在PK-15細(xì)胞上表達(dá)。 使用制備的抗CIITA4蛋白的多克隆抗體檢測(cè)轉(zhuǎn)染了PCDNA3.0-GFP-CIITA4的PK-15細(xì)胞中的CIITA4蛋白的表達(dá)情況,并且探索了表達(dá)CⅡTA4蛋白后對(duì)豬瘟病毒在PK-15細(xì)胞中的復(fù)制的影響,結(jié)果表明,表達(dá)CⅡTA4蛋白后,豬瘟病毒E2蛋白的表達(dá)沒有明顯變化,CⅡTA4蛋白在PK-15細(xì)胞中的表達(dá)與豬瘟病毒感染的相互關(guān)系還需進(jìn)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.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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本文編號(hào)：1692460