【作者】 尹业师；

【导师】 黄伟坚；

【作者基本信息】 广西大学 ， 预防兽医学， 2007， 硕士

【摘要】 参考GenBank上发表的猪圆环病毒Ⅱ型(porcine circovirus 2，PCV2)全基因组序列，设计一对引物，通过反向PCR技术扩增出广西、湛江、海南等地9株PCV2流行株的全基因，并进行了序列测定与分析，结果表明：9株PCV2的基因组全长为1768bp或1767bp，同源性比较发现，这9株PCV2核苷酸序列间同源性为95.6％～100％，与各国PCV2毒株的核苷酸同源性为95.1％-99.8％，其中与法国和新西兰代表株的亲缘关系较近，而与美国、加拿大和澳大利亚代表株的亲缘关系较远。9株PCV2的ORF2核苷酸及推导的氨基酸序列同源性分别为93.1％～100％和91.8％～99.6％，存在较大的变异。对2005年12月31日前GenBank上已公布的26株PCV1和262株PCV2的全基因序列进行了核苷酸的同源性比较，并对其ORF1和ORF2进行了核苷酸和推导氨基酸序列的比较，结果发现：从核苷酸水平来看，PCV1与PCV2明显分为两枝，PCV1间分布没有规律，而PCV2间又分为以来自美国、加拿大、澳大利亚的序列为代表的亚型Ⅰ和以来自法国和新西兰的序列为代表的亚型Ⅱ，但从ORF1和ORF2氨基酸水平来看，其分型并没有规律可循。对中国的97株PCV2核苷酸序列进行分析发现，来自广西、湛江、海南的9株和台湾的11株相对较集中，广西、湛江、海南的9株同属于亚型Ⅱ，台湾的11株同属于亚型Ⅰ，而来自其他各省的PCV2序列分布较复杂，大部分分布于亚型Ⅱ，少部分属于亚型Ⅰ。从遗传进化树可以看出，PCV2的分布并没有地域性和时序性。数据统计显示，尽管PCV2各毒株相隔近十年，但其同源性并没有发生太大的改变，可见PCV2相当保守。另外对PCV2的碱基和氨基酸突变位点进行统计发现，PCV2序列间变化更倾向于碱基的颠换，但无单个碱基和氨基酸的偏好性。猪2型圆环病毒(PCV2)是一种重要的免疫抑制性病原，近年来给养猪业造成了巨大的经济损失，但到目前为止，还没有找到有效的疫苗来预防此病的发生。本研究以PCDNA3为载体，与PCV2广西地方株的ORF2全序列、ORF2中的高变区V1、V2、V3、V1-V2连接，分别成功构建了真核表达质粒PCDNA3-ORF2、PCDNA3-V1、PCDNA3-V2、PCDNA3-V3和PCDNA3-V1-V2。对阳性真核表达质粒大量生产并纯化后，免疫5周龄左右的雌性小白鼠，每只200μg，然后分别于一免后7天、14天，二免后7天、14天用ELISA检测免疫后小白鼠血清中抗体的产生情况，结果发现这5种真核表达质粒均能刺激小白鼠产生PCV2特异性抗体，相比之下，真核表达质粒PCDNA3-V1-V2诱导小白鼠产生的PCV2抗体水平更高，维持时间更久。更多还原

【Abstract】 According to the sequence of porcine circovirus 2(PCV2)published in GenBank, a pair of primers were designed and local PCV2complete sequences were amplified by reverse PCR. The results showedthat the complete genome of the nine strains was 1768bp or 1767bpin length. There was a 95.6％～100％identity of the complete genomicnucleotides among the nine isolates and 95.1～99.8％identity withthe PCV2 isolates from GenBank. Comparison of nucleotide sequenceconfirmed that the nine isolates were close to the isolates fromFrance and Newsland. The nine isolates ORF2 had 93.1％～100％nucleotide sequence identidy and 91.8％～99.6％amino acid identidy.In this research, we analyzed nucleotide sequences of 26 strainsof PCV1 and 262 strains of PCV2, also, we analyzed the ORF1 and ORF2nucleotide sequences and deduced amino acid sequences. At thenucleotide level, the results show that PCV1 sequences and PCV2sequences are divided into two different branchs, obviously, and among the PCV1 sequences there haven’t any rules, but among PCV2sequences, there are divided into two different sub-gene types. Thesub-gene typeⅠis mainly consis of sequences from American, Canadaand Australia, and the sub-gene typeⅡis mainly consist of sequencesfrom Franch and Newzealand. At the amino acid level, the phylogenetictree is very complexity, and doesn’t have any rules. 97 strainssequences from china were analyzed, we find that 9 strains sequencesfrom GuangXi、ZhanJiang、HaiNan are part of sub-gene typeⅡand 11strains sequences from TAIWANG are part of sub-gene typeⅠ. But thedistributing of sequences from the other provinces of china are verycomplex, most of sequences are part of sub-gene typeⅡ, remains arepart of sub-gene typeⅠ. By statistic, we discover that, althoughnearby ten years have been pasted, the sequences homology of PCV don’t have large changes, the results showthat, PCV2 is very conserved.After analyzed the phylogenetic tree, we discover that thedistributing of PCV2 don’t have geographic and time restriction.Also, we analyzed the mutant dots of the PCV2 nucleotide sequencesand deduced amino acid sequences, we discover that, PCV2 nucleotidesequences are easy to transversion, but don’t have any tropism tobases or amino acids.Porcine circovirus type 2(PCV2) recently emerged as an importantinfectious pathogen for pigs in the world, moreover, PCV2 can induceimmunosuppression in postweaning pigs. But unfortunately, there is noefficient method or vaccine to control PCV2 infection until now. Inthis study, recombined plasmids PCDNA3-ORF2,PCDNA3-V1,PCDNA3-V2, PCDNA3-V3 and PCDNA3-V1-V2 were constructed by using PCDNA3, PCV2 ORF2, and variation regions of PCV2 ORF2.Production and purification of these plasmids, then immune the 5-weeks-old female mice, and detected the antibody in serum by usingELISA. The results show that all these plasmids can induce antibodyproduction in mice, but the level of antibody is difference, theplasmid PCDNA3-V1-V2 can induce higher level antibody and may enduremore long.更多还原