GGTA1基因敲除巴马小型猪SLA单倍型分型及转hEPCR基因猪的制备

发布时间：2018-04-20 12:58

本文选题：GGTA1基因 + 小型猪　；参考：《甘肃农业大学》2017年硕士论文

【摘要】：猪是人异种器官移植的理想供体,由α-1,3半乳糖苷(α-1,3Gal)引起的猪到人器官移植的超急性排斥反应已经基本解决,但是随之而来的细胞性排斥反应以及血栓和炎症成为目前亟待解决的问题,猪白细胞抗原(swine leukocyte antigen,SLA)是引起异种移植细胞性免疫排斥的主要因素之一,转人内皮蛋白C受体(endothelial cell protein C receptor,EPCR)可以有效降低猪到人器官移植引起的血栓和炎症。本研究主要探讨了α-1,3半乳糖苷转移酶(α-1,3-galactosyltransferase,GGTA1)基因敲除巴马小型猪(Bama minipig,BMP)的健康水平以及SLA等位基因和单倍型为异种器官移植研究筛选细胞排斥低的供体,制备可以降低异种器官移植引起的炎症和血栓反应的转hEPCR基因猪。本论文共有三部分研究内容,分别为:1.GGTA1基因敲除巴马小型猪的繁育及健康水平检测。本课题组制备的GGTA1基因敲除(α-1,3-galactosyltransferase knockout,GTKO)巴马小型猪已经繁育到F3代。本研究跟踪了GTKO巴马小型猪的遗传、窝产仔数和血常规血生化指标,评估其繁殖和健康状况。通过PCR产物测序鉴定仔猪GGTA1基因敲除类型,FITC-GSIB4与仔猪外周血单个核细胞(peripheral blood mononuclear cells,PBMC)孵育后流式细胞术检测α-1,3-Gal表达,以窝产仔数测定繁殖力,以血常规和血生化检测反映健康状况。结果显示,GGTA1基因的遗传符合孟德尔分离定律;GGTA1敲除纯合子(GGTA1-/-)仔猪的流式检测荧光强度低;GTKO巴马猪母猪头胎窝产仔数为7.38±1.64头,经产母猪窝产仔数为9.43±1.68头,与已有报道的普通巴马小型猪繁殖力无明显差异;血常规血生化检测的各项指标与普通巴马猪基本无差异。总之,连续3代GTKO巴马仔猪遗传稳定,繁殖力正常,生理健康。GTKO巴马小型猪可作为异种器官移植研究的可靠供体。2.GGTA1基因敲除巴马小型猪SLA单倍型分型。本研究主要对SLA基因上5个位点(SLA-1、SLA-2、SLA-3、SLA-DRB1和SLA-DQB1)进行研究。采用转录本PCR产物直接测序的方法获得GTKO巴马小型猪SLA等位基因和单倍型,补体依赖的淋巴细胞毒试验(Complementdependent cytotoxicity,CDC)检测不同SLA单倍型猪的PBMC与人血清反应的细胞死亡率。结果发现,GGTA1基因敲除巴马小型猪群的5个SLA位点共发现15个等位基因,其中SLA-2*05:07(序列号:KX022946)和SLA-3*03:10(序列号:KX022947)2条序列为新发现序列,GGTA1基因敲除巴马小型猪群共有5个SLA单倍型,分别为Hp-80.27、Hp-81.27、Hp-83a.45、Hp-82.44和Hp-83b.10c。4种杂合单倍型(Hp-80.27/81.27,Hp-82.44/83b.10c,Hp-80.27/83b.10c和Hp-83a.45/83b.10c)猪CDC结果细胞死亡率都小于27.5%,Hp-80.27/81.27和Hp-82.44/83b.10c单倍型的猪CDC结果细胞死亡率都小于17%。结果表明,不同SLA单倍型猪PBMC与人血清CDC试验结果不同,Hp-80.27/81.27和Hp-82.44/83b.10c单倍型的猪CDC结果细胞死亡率更低,更适合做异种器官移植研究供体。3.转hEPCR基因猪的制备。本研究构建广泛性表达启动子CAG调控的hEPCR基因表达载体pCAGGS-hEPCR-Puro,转染巴马五指山杂交猪胎儿成纤维细胞,通过体细胞克隆技术制备转基因克隆猪。利用PCR技术对克隆仔猪进行转基因鉴定,同时通过实时荧光定量PCR(qRT-PCR)、Western Blot及免疫组化方法分析hEPCR在转基因猪的各个器官中的表达。本研究成功构建了pCAGGS-hEPCR-Puro载体,转染细胞后筛选出71个有效克隆点,共得到2头转hEPCR阳性仔猪,qRT-PCR检测发现hEPCR基因在克隆仔猪肝脏、肾脏、心脏、脾脏、胰脏、肺脏、主动脉等主要器官都有表达,在主动脉、胰脏、肺脏中的表达水平较高。Western Blot和免疫组化结果表明hEPCR蛋白在耳朵、主动脉,胰脏、心脏和肾脏都有表达。本研究成功制备了转hEPCR基因猪,并且hEPCR基因在克隆猪的主要器官组织都有较高的表达水平,为异种器官移植研究制备了良好供体。
[Abstract]:Porcine is an ideal donor for xenotransplantation. The hyperacute rejection of porcine to human organ transplantation caused by alpha -1,3 galactoside (alpha -1,3Gal) has been basically solved, but the attendant cellular rejection and thrombus and inflammation are the problems to be solved urgently. The swine leukocyte antigen (SLA) is the leading factor. One of the main factors for xenograft rejection is that the endothelial cell protein C receptor (EPCR) C receptor (EPCR) can effectively reduce the thrombosis and inflammation caused by human organ transplantation. This study mainly discusses the alpha galactosidase (alpha -1,3-galactosyltransferase, GGTA1) gene knockout in Bama. The health level of Bama Minipig (BMP) and the SLA allele and haplotype for xenotransplantation screening the low cell rejection donor for the preparation of hEPCR transgenic pigs that can reduce the inflammatory and thrombus reaction caused by xenotransplantation. This paper has three parts: 1.GGTA1 gene knockout Bama miniature pig GGTA1 gene knockout (alpha -1,3-galactosyltransferase knockout, GTKO) Bama miniature pigs have been bred to the F3 generation. This study traced the inheritance, litter size and blood biochemical indexes of GTKO Bama miniature pigs, and evaluated their reproductive and health status. The offspring were sequenced and identified by PCR products. GGTA1 gene knockout type, FITC-GSIB4 and peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) of piglets were incubated by flow cytometry to detect the expression of alpha -1,3-Gal. The fecundity was measured by litter size, and the health status was reflected by blood routine and blood biochemical tests. The results showed that the inheritance of GGTA1 gene conforms to the Mendel law of separation. The flow detection fluorescence intensity of GGTA1 knockout homozygote (GGTA1-/-) piglets was low, the number of litter size in the head litter of GTKO Bama sows was 7.38 + 1.64, the number of litter size of the sows was 9.43 + 1.68 heads, and there was no significant difference from the common Bama miniature pig's fecundity, and the indexes of blood routine blood biochemistry were basically no different from that of normal Bama pigs. In a word, the 3 generations of GTKO Bama piglets have been genetically stable, and the reproductive capacity is normal. The physiological healthy.GTKO Bama miniature pig can be used as a reliable donor.2.GGTA1 gene for SLA haplotyping of the Bama miniature pig. This study mainly studies the 5 loci of the SLA gene (SLA-1, SLA-2, SLA-3, SLA-DRB1 and SLA-DQB1). The GTKO Bama miniature pig SLA allele and haplotype were obtained by direct sequencing of the PCR product, and the complement dependent lymphocyte toxicity test (Complementdependent cytotoxicity, CDC) was used to detect the cell death rate of PBMC and human serum reaction in different SLA haplotype pigs. The results showed that the GGTA1 gene knocked out 5 SLA loci of the Bama miniature pig. 15 alleles were found, including 2 sequences of SLA-2*05:07 (sequence number: KX022946) and SLA-3*03:10 (sequence number: KX022947). There were 5 SLA haplotypes of GGTA1 gene knockout Bama miniature pigs, Hp-80.27, Hp-81.27, Hp-83a.45, Hp-82.44 and Hp-83b.10c.4 hybrid haplotypes. 80.27/83b.10c and Hp-83a.45/83b.10c) the mortality of pig CDC cells was less than 27.5%. The results of CDC results in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotype pigs were less than that of 17%., and the results of PBMC in different SLA haplotype pigs were different from those of CDC in human serum, and the result cells died in Hp-80.27/81.27 and Hp-82.44/83b.10c haplotypes. The death rate is lower, and it is more suitable for the preparation of the donor.3. transgenic hEPCR gene pig. This study constructs a hEPCR gene expression vector pCAGGS-hEPCR-Puro regulated by the promoter CAG, which is widely used to transfect the fetal fibroblasts of Bama Five Fingers Group hybrid pig, and the transgenic cloned pig is prepared by somatic cell clon technology. The PCR technology is used. The cloning of piglets was genetically modified, and the expression of hEPCR in various organs of transgenic pigs was analyzed by real-time quantitative PCR (qRT-PCR), Western Blot and immunohistochemical method. The pCAGGS-hEPCR-Puro vector was successfully constructed. After transfecting the cells, 71 effective clones were screened and 2 hEPCR positive piglets were obtained, qRT-PCR was obtained. The expression of hEPCR gene in the liver, kidney, kidney, heart, spleen, pancreas, pancreas, lung and aorta of piglets was expressed. The high expression level of.Western Blot and immunohistochemistry in aorta, pancreas and lung showed that hEPCR protein was expressed in the ear, active vein, pancreas, heart and kidney. Transgenic hEPCR pigs and hEPCR genes have higher expression level in the main organs and tissues of the cloned pigs, and have prepared good donors for xenotransplantation.

【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R617;Q78

【参考文献】