稳定共表达A2单倍型鸭TAP1和TAP2蛋白细胞系的建立

发布时间：2018-05-07 04:04

本文选题：鸭 + TAP1　；参考：《中国农业科学院》2015年硕士论文

【摘要】：抗原处理相关转运体(Transporter associated with antigen processing,TAP)属ABC(ATP-binding cassette,ABC)转运器家族,其编码基因TAP1和TAP2均位于主要组织相容性复合体(Major histocompatibility complex,MHC)。TAP1和TAP2编码的蛋白组成二聚体。在不同种属动物体内,TAP1和TAP2在MHC上的具体位置不同。TAP在多种伴侣分子的协同下,包括钙网蛋白、钙联蛋白、氧化还原酶ERp57和TAP相关蛋白(tapasin)等,将胞浆内被蛋白酶体降解的内源性抗原肽转运至内质网,与MHC I类分子结合,所形成的MHC-肽复合物通过高尔基体被提呈到细胞表面,引起细胞毒性T细胞的免疫应答。TAP蛋白的缺失和突变会影响抗原肽的提呈,最终损害免疫应答功能。本研究采用RT-PCR技术从HBK-SPF鸭脾脏中扩增得到TAP1和TAP2基因,序列分析表明TAP2基因的多态性高于TAP1。利用单核苷酸多态性(SNP)分子标记技术,对正在选育的第3代单倍型鸭的TAP2基因型进行遗传稳定性监测,结果表明单倍型鸭的MHC型保持稳定。经过序列比对,A2品系的SNP最多,进一步对A2品系的TAP1和TAP2基因进行了生物信息学分析。将A2单倍型鸭的TAP1(A2TAP1)全部编码区和A2 TAP2的肽结合区分别克隆至p ET-30a(+),成功构建了原核表达质粒p ET-A2TAP1和p ET-A2TAP2PBD,并转化至大肠杆菌BL21(DE3)中经IPTG诱导表达,获得以包涵体形式存在的特异性条带,大小分别约为51 k Da和22 k Da重组蛋白。重组蛋白免疫BALB/c小鼠,制备并纯化了鼠多抗血清。将编码A2TAP1和A2TAP2的全基因同时克隆到真核表达载体p IRES中,获得真核表达质粒p IRES-A2TAP1-A2TAP2。利用Amaxa Cell Line Nucleofector Kit L将质粒电转染至小鼠淋巴细胞系RMA-S细胞,G418对细胞进行抗性筛选,最终获得稳定表达A2TAP1蛋白和A2TAP2蛋白的RMA-S细胞系RMA-S-A2TAP1-A2TAP2。对第30代细胞系进行RT-PCR检测、间接免疫荧光抗体试验和蛋白免疫印迹鉴定,表明蛋白A2TAP1和A2TAP2在RMA-S细胞中得到共表达,并具有免疫学活性。本研究为在细胞水平上研究鸭TAP生物学功能提供了物质条件。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S834

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