本文選題：鴨 + TAP1��； 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：抗原處理相關(guān)轉(zhuǎn)運(yùn)體(Transporter associated with antigen processing,TAP)屬ABC(ATP-binding cassette,ABC)轉(zhuǎn)運(yùn)器家族,其編碼基因TAP1和TAP2均位于主要組織相容性復(fù)合體(Major histocompatibility complex,MHC)。TAP1和TAP2編碼的蛋白組成二聚體。在不同種屬動物體內(nèi),TAP1和TAP2在MHC上的具體位置不同。TAP在多種伴侶分子的協(xié)同下,包括鈣網(wǎng)蛋白、鈣聯(lián)蛋白、氧化還原酶ERp57和TAP相關(guān)蛋白(tapasin)等,將胞漿內(nèi)被蛋白酶體降解的內(nèi)源性抗原肽轉(zhuǎn)運(yùn)至內(nèi)質(zhì)網(wǎng),與MHC I類分子結(jié)合,所形成的MHC-肽復(fù)合物通過高爾基體被提呈到細(xì)胞表面,引起細(xì)胞毒性T細(xì)胞的免疫應(yīng)答。TAP蛋白的缺失和突變會影響抗原肽的提呈,最終損害免疫應(yīng)答功能。本研究采用RT-PCR技術(shù)從HBK-SPF鴨脾臟中擴(kuò)增得到TAP1和TAP2基因,序列分析表明TAP2基因的多態(tài)性高于TAP1。利用單核苷酸多態(tài)性(SNP)分子標(biāo)記技術(shù),對正在選育的第3代單倍型鴨的TAP2基因型進(jìn)行遺傳穩(wěn)定性監(jiān)測,結(jié)果表明單倍型鴨的MHC型保持穩(wěn)定。經(jīng)過序列比對,A2品系的SNP最多,進(jìn)一步對A2品系的TAP1和TAP2基因進(jìn)行了生物信息學(xué)分析。將A2單倍型鴨的TAP1(A2TAP1)全部編碼區(qū)和A2 TAP2的肽結(jié)合區(qū)分別克隆至p ET-30a(+),成功構(gòu)建了原核表達(dá)質(zhì)粒p ET-A2TAP1和p ET-A2TAP2PBD,并轉(zhuǎn)化至大腸桿菌BL21(DE3)中經(jīng)IPTG誘導(dǎo)表達(dá),獲得以包涵體形式存在的特異性條帶,大小分別約為51 k Da和22 k Da重組蛋白。重組蛋白免疫BALB/c小鼠,制備并純化了鼠多抗血清。將編碼A2TAP1和A2TAP2的全基因同時克隆到真核表達(dá)載體p IRES中,獲得真核表達(dá)質(zhì)粒p IRES-A2TAP1-A2TAP2。利用Amaxa Cell Line Nucleofector Kit L將質(zhì)粒電轉(zhuǎn)染至小鼠淋巴細(xì)胞系RMA-S細(xì)胞,G418對細(xì)胞進(jìn)行抗性篩選,最終獲得穩(wěn)定表達(dá)A2TAP1蛋白和A2TAP2蛋白的RMA-S細(xì)胞系RMA-S-A2TAP1-A2TAP2。對第30代細(xì)胞系進(jìn)行RT-PCR檢測、間接免疫熒光抗體試驗和蛋白免疫印跡鑒定,表明蛋白A2TAP1和A2TAP2在RMA-S細(xì)胞中得到共表達(dá),并具有免疫學(xué)活性。本研究為在細(xì)胞水平上研究鴨TAP生物學(xué)功能提供了物質(zhì)條件。
[Abstract]:The antigen processing related transporter (Transporter associated with antigen processing, TAP) belongs to the ABC (ATP-binding cassette, ABC) transporter family. The encoding gene TAP1 and TAP2 are located in the main histocompatibility complex and the proteins encoded by the two polymers. In different species of the animal body Inside, the specific location of TAP1 and TAP2 on MHC is different from.TAP, which includes calcium reticulin, calcalin, oxidoreductase ERp57 and TAP related protein (tapasin), and transtransport endogenous antigen peptides degraded by proteasome to endoplasmic reticulum, and MHC I molecules, and the MHC- peptide complex formed by MHC. Golgi Ti was presented to the cell surface, causing the deletion and mutation of the immune response.TAP protein of the cytotoxic T cells to affect the presentation of the antigen peptide and ultimately damage the immune response function. The RT-PCR technique was used to amplify the TAP1 and TAP2 genes from the spleen of HBK-SPF duck. The sequence analysis showed that the polymorphism of the TAP2 gene was higher than that of the TAP1. benefit. A single nucleotide polymorphism (SNP) molecular marker technique was used to monitor the genetic stability of the TAP2 genotypes of the third generation haplotype ducks that were being selected. The results showed that the MHC type of the haplotype ducks remained stable. After sequence alignment, the SNP of the A2 strain was the most, and the TAP1 and TAP2 genes of the A2 strain were further analyzed. The whole coding region of TAP1 (A2TAP1) of type ducks and the peptide binding region of A2 TAP2 were cloned to P ET-30a (+) respectively. The prokaryotic expression plasmid P ET-A2TAP1 and P ET-A2TAP2PBD were successfully constructed and transformed into the Escherichia coli BL21 (DE3) to be induced and expressed. The specific bands in the inclusion body form were obtained, the size of which was about 51 and 22 of the recombinant eggs, respectively. The recombinant protein was immunized with BALB/c mice, and the rat polyclonal antibody was prepared and purified. The whole gene encoding A2TAP1 and A2TAP2 was cloned into the eukaryotic expression vector p IRES, and the eukaryotic expression plasmid P IRES-A2TAP1-A2TAP2. was transfected to the mouse lymphocyte clone cells by Amaxa Cell Line Nucleofector Kit. The cell line was screened for resistance, and the RMA-S cell line that stably expressed A2TAP1 protein and A2TAP2 protein was obtained by RT-PCR detection, indirect immunofluorescence antibody test and protein immunoblotting identification, which showed that protein A2TAP1 and A2TAP2 were co expressed in RMA-S cells and had immunological activity. The study provided material conditions for studying the biological function of duck TAP at cellular level.

【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S834

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本文編號：1855309