本文選題：犬副流感病毒　切入點(diǎn)：微型基因組　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：犬副流感病毒(canine parainfluenza virus,CPIV)是引起犬的自限性氣管支氣管炎,犬窩咳的主要病因。臨床上,CPIV單獨(dú)感染不引起致死性疾病,只表現(xiàn)為溫和的呼吸道癥狀,而這種情況并不常見;常見的是CPIV與其他病原的混合感染,使病情加劇,大幅度提高死亡率。自1967年首次分離得到該病毒以來,世界各地陸續(xù)有相關(guān)報道,目前該病毒感染范圍已波及全世界,在我國亦出現(xiàn)了普遍流行趨勢。在各類病毒疫苗的開發(fā)研究中,最為重大的科學(xué)進(jìn)展是反向遺傳技術(shù)。目前,CPIV的基因工程疫苗尚未見報道,但對人副流感病毒(human parainfluenza virus,HPIV)和牛副流感病毒3型(Bovine parainfluenza virus type3,BPIV3)已有較多的研究。隨著負(fù)鏈RNA病毒反向遺傳學(xué)技術(shù)的日趨成熟,副流感病毒(parainfluenza virus,PIV)作為活病毒表達(dá)載體方面有廣闊的應(yīng)用前景,HPIV和BPIV3的現(xiàn)有研究成果對CPIV的研究有借鑒意義。本實(shí)驗(yàn)室以構(gòu)建CPIV的感染性分子克隆為目的。根據(jù)Gen Bank(KC237064.1)中PIV5 1168-1毒株序列設(shè)計引物,應(yīng)用RT-PCR法對購自ATCC的CPIV HRB-V病毒株分段擴(kuò)增,克隆到pHW2000載體中并進(jìn)行測序,經(jīng)序列拼接獲得全基因組序列。與Gen Bank登錄的PIV代表毒株進(jìn)行對比分析�；蛐蛄蟹治霰砻�,HRB-V株基因組全長15246nt,含5'-末端、3'-末端和7個ORF,具有PIV基因組結(jié)構(gòu)的典型特征;將結(jié)構(gòu)蛋白F、HN以及全基因序列與參考毒株進(jìn)行比較分析并做遺傳進(jìn)化樹,結(jié)果表明:HRB-V與CPI-和CPI+屬同一分支,親緣性相近。同源性分析結(jié)果表明:HRB-V與各病毒株的F基因的核苷酸同源性在96.5%~98.8%之間,氨基酸同源性為94.2%~98%;HN基因的核苷酸同源性在98.1%~99.5%之間,氨基酸同源性為97%~99.3%。本研究系統(tǒng)了解了CPIV的基因組結(jié)構(gòu)及遺傳進(jìn)化特點(diǎn)。參考已知的HRB-V病毒株序列設(shè)計引物,RT-PCR擴(kuò)增NP、P和L基因,分別克隆入真核表達(dá)載體pCI-neo中,構(gòu)建輔助質(zhì)粒;同時使eGFP取代病毒整個編碼區(qū),只保留兩個末端與病毒復(fù)制、轉(zhuǎn)錄和病毒粒子包裝相關(guān)的調(diào)控序列,將其反向克隆入轉(zhuǎn)錄載體pVAX1中,構(gòu)建該病毒的微型基因組質(zhì)粒,應(yīng)用此微型基因組對輔助質(zhì)粒的表達(dá)產(chǎn)物進(jìn)行功能鑒定。結(jié)果表明NP、P和L三種蛋白的正常表達(dá)并發(fā)揮RNA聚合酶作用,為病毒拯救奠定基礎(chǔ)。本研究成功構(gòu)建了CPIV的分子克隆,并進(jìn)行系統(tǒng)分析;同時構(gòu)建了病毒輔助質(zhì)粒及微型基因組,鑒定了輔助質(zhì)粒的RNA聚合酶功能。以上研究為實(shí)驗(yàn)室構(gòu)建CPIV反向遺傳操作技術(shù)平臺奠定基礎(chǔ)。
[Abstract]:Canine parainfluenza virus (CPIV) is the main cause of canine self-limited tracheobronchial bronchitis and burrow cough in dogs. Clinical infection of canine parainfluenza virus alone does not cause fatal diseases, but shows mild respiratory symptoms, which is not common. It is common to see a mixture of CPIV and other pathogens, which exacerbates the disease and dramatically increases the mortality rate. Since the virus was first isolated in 1967, there have been reports from around the world that the virus has spread to the whole world. The most important scientific progress in the development and research of various viral vaccines is reverse genetic technology. At present, the genetic engineering vaccine of CPIV has not been reported. However, more studies have been done on human parainfluenza virus (HPIV) and bovine parainfluenza virus type 3 (BPIV3). Parainfluenza virus (PIV) as a living virus expression vector has a broad application prospect. The existing research results of HPIV and BPIV3 can be used for reference in the study of CPIV. The purpose of our laboratory is to construct the infectious molecular clone of CPIV. According to Gen. The sequence of PIV5 1168-1 strain in KC237064.1) was designed. The CPIV HRB-V virus strain purchased from ATCC was amplified by RT-PCR, cloned into pHW2000 vector and sequenced. The whole genome sequence was obtained by sequence splicing, and compared with the PIV representative strain registered by Gen Bank. The gene sequence analysis showed that the whole genome of HRBV strain was 15246nt in length, which contained 5H-terminal 3 terminal and 7 ORFs, and had the typical characteristics of PIV genomic structure. The structure protein FHN and the whole gene sequence were compared with the reference strain and the genetic evolution tree was made. The results showed that the two species belong to the same branch as CPI- and CPI. The results of homology analysis showed that the nucleotide homology of F gene and amino acid homology were 96.5% and 98.8%, respectively, and the nucleotide homology of HN gene was 98.1% and 99.5%, respectively. The amino acid homology was 970.In this study, the genomic structure and genetic evolution characteristics of CPIV were studied. The nucleotide sequences of HRB-V virus were amplified by RT-PCR and cloned into eukaryotic expression vector pCI-neo, respectively, and the auxiliary plasmids were constructed. At the same time, eGFP was used to replace the whole coding region of the virus, and only two regulatory sequences related to virus replication, transcription and virus particle packaging were retained, and then cloned into the transcriptional vector pVAX1 to construct the microgenome plasmid of the virus. The microgenome was used to identify the function of the expression product of the auxiliary plasmid. The results showed that the three proteins expressed normally and played the role of RNA polymerase, which laid the foundation for virus rescue. In this study, the molecular cloning of CPIV was successfully constructed. At the same time, the virus auxiliary plasmid and microgenome were constructed, and the function of RNA polymerase of the auxiliary plasmid was identified, which laid a foundation for the laboratory construction of CPIV reverse genetic manipulation platform.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.655

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