【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的急性、烈性傳染病,主要感染牛、羊及豬等偶蹄動(dòng)物�？谔阋吡餍械貐^(qū),滅活疫苗免疫是防控疫情的關(guān)鍵措施,而滅活疫苗要發(fā)揮作用的關(guān)鍵,是確保FMDV顆粒的完整,即146S粒子的完整。然而146S粒子穩(wěn)定性較差,溫度升高或pH發(fā)生變化時(shí)會(huì)使146S分解為12S粒子,導(dǎo)致滅活疫苗免疫失敗。于是研究者們根據(jù)146S粒子分子結(jié)構(gòu)的研究成果,嘗試通過(guò)氨基酸殘基替換來(lái)提升FMDV的酸、熱穩(wěn)定性,進(jìn)而為篩選候選疫苗毒株提供技術(shù)支持。本研究在先前的研究基礎(chǔ)上,選取了疫苗種毒O/GX/09-7毒株的結(jié)構(gòu)蛋白VP1的17位及VP2的93位氨基酸殘基作為替換位點(diǎn)。利用設(shè)計(jì)的突變引物,成功擴(kuò)增出含堿基突變的O/GX/09-7毒株P(guān)1區(qū)序列。成功將目的片段插入到T載體,構(gòu)建8種克隆質(zhì)粒,成功將目的片段插入到本實(shí)驗(yàn)室已構(gòu)建好的FMDV載體p43-CHA90-Vector上,構(gòu)建出8種重組質(zhì)粒。利用8種重組質(zhì)粒及感染性克隆pOZK/93轉(zhuǎn)錄出病毒基因組RNA并且轉(zhuǎn)染進(jìn)入BHK-21細(xì)胞中,成功拯救出2株病毒vOZK/93和vOS93H。對(duì)病毒vOZK/93和vOS93H及親本毒株O/GX/09-7分別進(jìn)行酸、熱處理,測(cè)量處理后病毒的TCID50的值及146S粒子含量,并與空白對(duì)照進(jìn)行比較,研究氨基酸替換對(duì)O/GX/09-7毒株穩(wěn)定性的影響。結(jié)果顯示,vOZK/93和vOS93H兩株病毒的熱穩(wěn)定性均與O/GX/09-7毒株相似,沒(méi)有明顯變化。病毒vOZK/93的酸穩(wěn)定性與O/GX/09-7毒株相似,但突變病毒vOS93H的酸穩(wěn)定性與O/GX/09-7毒株相比,有一定的提高,初步達(dá)到試驗(yàn)預(yù)期目的。
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is an acute and severe infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV), which mainly affects cloven-hoofed animals such as cattle, sheep and pigs. In the epidemic areas of foot-and-mouth disease, inactivated vaccine immunization is the key measure to prevent and control the epidemic situation, and the key of inactivated vaccine to play a role is to ensure the integrity of FMDV particles, that is, the integrity of 146s particles. However, 146S particles have poor stability. When the temperature increases or pH changes, 146S particles will be decomposed into 12s particles, resulting in the inactivated vaccine immune failure. Therefore, according to the molecular structure of 146S particles, the researchers try to improve the acid and thermal stability of FMDV by amino acid residues substitution, and then provide technical support for screening candidate vaccine strains. On the basis of previous studies, the 17 site of structural protein VP1 and 93 amino acid residue of VP2 of O/GX/09-7 strain were selected as substitution sites. The P1 region of O/GX/09-7 strain containing base mutation was amplified by using the designed primers. The target fragment was successfully inserted into T vector to construct eight kinds of cloning plasmids. The target fragment was successfully inserted into the FMDV vector p43-CHA90-Vector which had been constructed in our laboratory, and eight recombinant plasmids were constructed. Eight recombinant plasmids and infectious clone pOZK/93 were used to transcribe the genomic RNA of the virus and transfect it into BHK-21 cells. Two strains of virus vOZK/93 and vOS93H. were successfully rescued. The virus vOZK/93, vOS93H and parent strain O/GX/09-7 were treated with acid and heat treatment respectively. The TCID50 value and 146s particle content of the virus were measured and compared with the blank control. The effect of amino acid substitution on the stability of O/GX/09-7 strain was studied. The results showed that the thermal stability of both vOZK/93 and vOS93H strains was similar to that of O/GX/09-7 strain, but there was no significant change. The acid stability of the virus vOZK/93 was similar to that of the O/GX/09-7 strain, but the acid stability of the mutant virus vOS93H was higher than that of the O/GX/09-7 strain.
【學(xué)位授予單位】：中國(guó)獸醫(yī)藥品監(jiān)察所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.65

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