本文選題：羊口瘡病毒　切入點(diǎn)：B2L蛋白　出處：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：羊傳染性膿皰病(Contagious Ecthyma,CE)俗稱羊口瘡,是由痘病毒科(Poxviridae)副痘病毒屬(Parapoxvirus)的羊口瘡病毒(Orf virus,ORFV)引起的一種接觸性皮膚傳染病,主要感染綿羊和山羊等小反芻動(dòng)物,有時(shí)甚至?xí)腥救祟?是全球性廣泛分布的病毒性傳染病之一。由于羊口瘡對(duì)于農(nóng)牧業(yè)的潛在威脅以及存在人畜共患風(fēng)險(xiǎn),因此,ORFV及其致病因子的研究已經(jīng)成為熱點(diǎn)。其中,B2L基因作為不可替代的關(guān)鍵基因,始終被研究者密切關(guān)注。該基因位于病毒基因組中央高度保守區(qū)域,是病毒一種重要的保護(hù)性抗原基因,其編碼的病毒粒子囊膜部分蛋白作為主要的抗原可以刺激機(jī)體產(chǎn)生強(qiáng)烈的免疫反應(yīng),并活化淋巴細(xì)胞;同時(shí),B2L對(duì)于病毒的裝配、擴(kuò)散和侵染可能十分必要。然而,針對(duì)B2L的研究目前僅僅局限于核酸鑒定,并未有深入研究其功能的報(bào)道。因此,可以采用新型基因編輯方法加以驗(yàn)證。首先,為了驗(yàn)證ORFV B2L基因被編輯前后的功能,針對(duì)B2L N端構(gòu)象表位的單克隆抗體2E4,在分子水平上驗(yàn)證2E4抗體輕、重鏈可變區(qū)序列中互補(bǔ)決定區(qū)(complementarity determining regions,CDRs)與B2L表位的分子結(jié)構(gòu)對(duì)應(yīng)性。通過體外擴(kuò)增可變區(qū)序列和測(cè)序,并上傳NCBI-Ig BLAST獲得2E4抗體可變區(qū)的CDRs和骨架區(qū)(framework regions,FRs)氨基酸結(jié)構(gòu)。繼而,采用SWISS-MODEL同源建模方法,展示抗體分子輕、重鏈可變區(qū)的三維空間構(gòu)型,并模擬了可能的ORFV B2L抗原表位多肽結(jié)合部位(Pocket)。通過CDRs區(qū)氨基酸突變驗(yàn)證了其與B2L抗原結(jié)合的重要性。其二,采用CRISPR/Cas9基因編輯技術(shù)對(duì)ORFV B2L基因分別在體外和體內(nèi)進(jìn)行編輯。通過設(shè)計(jì)特異的打靶g(shù) RNA序列并體外轉(zhuǎn)錄成單鏈m RNA(sg RNA),在Cas9核酸酶作用下對(duì)B2L-p ET-32a重組質(zhì)粒進(jìn)行切割,從而驗(yàn)證g RNA序列的選取可行性。隨后將PMJ920和g RNA1-p GEMT-easy質(zhì)粒共轉(zhuǎn)染于HEK293細(xì)胞,利用病毒蝕斑純化技術(shù)對(duì)編輯后病毒變異株進(jìn)行純化和擴(kuò)大培養(yǎng),通過測(cè)序分析B2L基因編輯情況。測(cè)序結(jié)果顯示,篩選出的1株病毒在PAM序列(CGG)5’端第5個(gè)堿基處,由原來的胞嘧啶(C)突變?yōu)轼B嘌呤(G),但氨基酸依然是絲氨酸(S);另1株病毒變異株(命名為OV_HLJ-ΔB2L)在PAM序列(CGG)5’端第4個(gè)堿基開始連續(xù)缺失4個(gè)堿基序列,導(dǎo)致后面的氨基酸序列移碼并提前有終止子出現(xiàn)。通過間接免疫熒光證實(shí)病毒變異株OV_HLJ-ΔB2L不能與2E4單抗結(jié)合。將OV_HLJ-ΔB2L變異株接種動(dòng)物羊后,其致病力減弱。本研究采用同源建模方法,展示抗體分子輕、重鏈可變區(qū)的CDRs,并模擬了可能的B2L表位多肽結(jié)合部位,采用點(diǎn)突變法證明了表位與CDRs結(jié)合的特異性。采用CRISPR/Cas9技術(shù)對(duì)ORFV B2L基因進(jìn)行編輯,并篩選出缺失B2L基因的病毒變異株OV_HLJ-ΔB2L,為今后B2L基因的應(yīng)用和研究ORFV其它的功能性基因奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.654

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