羊口疮病毒单抗2E4可变区同源建模及病毒B2L基因体外编辑的研究

发布时间：2018-03-10 08:51

本文选题：羊口疮病毒　切入点：B2L蛋白　出处：《黑龙江八一农垦大学》2017年硕士论文　论文类型：学位论文

【摘要】：羊传染性脓疱病(Contagious Ecthyma,CE)俗称羊口疮,是由痘病毒科(Poxviridae)副痘病毒属(Parapoxvirus)的羊口疮病毒(Orf virus,ORFV)引起的一种接触性皮肤传染病,主要感染绵羊和山羊等小反刍动物,有时甚至会感染人类,是全球性广泛分布的病毒性传染病之一。由于羊口疮对于农牧业的潜在威胁以及存在人畜共患风险,因此,ORFV及其致病因子的研究已经成为热点。其中,B2L基因作为不可替代的关键基因,始终被研究者密切关注。该基因位于病毒基因组中央高度保守区域,是病毒一种重要的保护性抗原基因,其编码的病毒粒子囊膜部分蛋白作为主要的抗原可以刺激机体产生强烈的免疫反应,并活化淋巴细胞;同时,B2L对于病毒的装配、扩散和侵染可能十分必要。然而,针对B2L的研究目前仅仅局限于核酸鉴定,并未有深入研究其功能的报道。因此,可以采用新型基因编辑方法加以验证。首先,为了验证ORFV B2L基因被编辑前后的功能,针对B2L N端构象表位的单克隆抗体2E4,在分子水平上验证2E4抗体轻、重链可变区序列中互补决定区(complementarity determining regions,CDRs)与B2L表位的分子结构对应性。通过体外扩增可变区序列和测序,并上传NCBI-Ig BLAST获得2E4抗体可变区的CDRs和骨架区(framework regions,FRs)氨基酸结构。继而,采用SWISS-MODEL同源建模方法,展示抗体分子轻、重链可变区的三维空间构型,并模拟了可能的ORFV B2L抗原表位多肽结合部位(Pocket)。通过CDRs区氨基酸突变验证了其与B2L抗原结合的重要性。其二,采用CRISPR/Cas9基因编辑技术对ORFV B2L基因分别在体外和体内进行编辑。通过设计特异的打靶g RNA序列并体外转录成单链m RNA(sg RNA),在Cas9核酸酶作用下对B2L-p ET-32a重组质粒进行切割,从而验证g RNA序列的选取可行性。随后将PMJ920和g RNA1-p GEMT-easy质粒共转染于HEK293细胞,利用病毒蚀斑纯化技术对编辑后病毒变异株进行纯化和扩大培养,通过测序分析B2L基因编辑情况。测序结果显示,筛选出的1株病毒在PAM序列(CGG)5’端第5个碱基处,由原来的胞嘧啶(C)突变为鸟嘌呤(G),但氨基酸依然是丝氨酸(S);另1株病毒变异株(命名为OV_HLJ-ΔB2L)在PAM序列(CGG)5’端第4个碱基开始连续缺失4个碱基序列,导致后面的氨基酸序列移码并提前有终止子出现。通过间接免疫荧光证实病毒变异株OV_HLJ-ΔB2L不能与2E4单抗结合。将OV_HLJ-ΔB2L变异株接种动物羊后,其致病力减弱。本研究采用同源建模方法,展示抗体分子轻、重链可变区的CDRs,并模拟了可能的B2L表位多肽结合部位,采用点突变法证明了表位与CDRs结合的特异性。采用CRISPR/Cas9技术对ORFV B2L基因进行编辑,并筛选出缺失B2L基因的病毒变异株OV_HLJ-ΔB2L,为今后B2L基因的应用和研究ORFV其它的功能性基因奠定了基础。
[Abstract]:Contagious Ecthymae, commonly known as sheep mouth sore, is a contact skin infection caused by the parapoxviridaevirus (Parapoxviridaeus), which mainly infects small ruminants, such as sheep and goats, and sometimes even human beings. Is one of the most widespread viral infectious diseases in the world. Due to the potential threat of sheep and mouth sores to agriculture and animal husbandry and the risk of zoonosis, Therefore, the study of ORFV and its pathogenic factors has become a hot topic, among which the B2L gene, as an irreplaceable key gene, has always been paid close attention to by researchers. It is located in the highly conserved region of the virus genome. Virus is an important protective antigen gene, which encodes part of the virus particle envelope protein as the main antigen can stimulate the body to produce a strong immune response, and activate lymphocytes; at the same time, B2L to the assembly of the virus, Diffusion and infection may be necessary. However, the study of B2L is limited to nucleic acid identification, and there are no reports of its function. Therefore, a new gene editing method can be used to verify it. In order to verify the function of ORFV B2L gene before and after editing, the monoclonal antibody 2E4 against the N-terminal conformation epitope of B2L was confirmed to be light at molecular level. The molecular structure of B2L epitopes in the complementary determining determining regions of heavy chain variable region sequences was compared. The CDRs and skeleton regions of the variable region of 2E4 antibody were amplified and sequenced in vitro, and the amino acid structure of the variant region of 2E4 antibody was obtained by uploading NCBI-Ig BLAST, and then the amino acid structure of the variable region of 2E4 antibody was obtained. Using SWISS-MODEL homology modeling method, the three-dimensional configuration of light and heavy chain variable region of antibody molecules was demonstrated. The potential epitope peptide binding site of ORFV B2L was simulated. The importance of binding to B2L antigen was verified by amino acid mutation in CDRs region. The ORFV B2L gene was edited in vitro and in vivo by CRISPR/Cas9 gene editing technique. The recombinant plasmid of B2L-p ET-32a was dissected by designing specific targeting g RNA sequence and transcribing into single strand RNA(sg RNAs in vitro. Then the PMJ920 and g RNA1-p GEMT-easy plasmids were co-transfected into HEK293 cells, and the modified mutant was purified and cultured by virus plaque purification technique. The sequence analysis of B2L gene showed that the selected strain of the virus was at the fifth base at the 5 'terminal of the PAM sequence. The original cytosine cytosine C was mutated to guanine guanine, but the amino acid was still serine, and another variant strain (named OVHLJ- 螖 B2L) began to lose 4 base sequences at the 5th 'end of the PAM sequence, which was named OVHLJ- 螖 B2L. It was confirmed by indirect immunofluorescence that the virus strain OVHLJ- 螖 B2L could not bind to 2E4 monoclonal antibody. After inoculating OVALJ- 螖 B2L variant strain with animal sheep, In this study, we used homologous modeling method to display CDRswith light and heavy chain variable region of antibody molecules, and to simulate the potential binding sites of B2L epitope peptides. The specificity of epitope binding to CDRs was proved by point mutation method. ORFV B2L gene was edited by CRISPR/Cas9. The virus variant OVHLJ- 螖 B2L with B2L gene deletion was screened, which laid a foundation for the application of B2L gene and the study of other functional genes in ORFV.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.654

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