摘要
猪圆环病毒Ⅱ型(Porcine Circovirus Type2, PCV-2)是引起断奶仔猪多系统衰竭综合征、母猪繁殖障碍、猪皮炎肾病综合征和呼吸道综合征等相关疾病的重要病原。PCV-2主要损害猪的免疫功能,造成免疫系统全面衰竭,进而继发感染其他疾病,给养猪业带来巨大的损失。猪细小病毒(Porcine Parvovirus, PPV)作为猪繁殖障碍的主要病原体之一,主要引起怀孕母猪流产、死产及胎儿木乃伊化,国内外相继报道猪圆环病毒和猪细小病毒混合感染的病例,使养猪业面临着严峻的挑战。
本研究在PK-15细胞上增殖猪圆环病毒Ⅱ型分离株(PCV-2)和猪细小病毒7909株(PPV),收获的细胞毒采用蛋白酶K法提取两种病毒的全病毒核酸。利用primer5.0软件自行设计了两对特异性引物并扩增出ORF2和VP2的全基因。然后选取HindⅢ、BamHⅠ和EcoRⅤ三个酶切,将两段目的基因分别连接到pCDNA3.1(+)真核表达载体,构建了三种重组质粒pCDNA3.1-ORF2、pCDNA3.1-VP2和pCDNA3.1-ORF2-VP2,通过酶切,10 g/L的琼脂糖凝胶电泳检测,结果表明:ORF2和VP2正确的插入到载体中。
采用碱裂解法和聚乙二醇沉淀法大量提取三种重组质粒,利用紫外分光光度计分析质粒浓度及纯度,结果显示:重组质粒浓度较高,并且没有蛋白污染。对试验小鼠左后肢股四头肌进行盐酸普鲁卡因药物预处理,预处理24h后,同样的部位进行重组质粒肌肉注射,免疫剂量为0.2mg/只,共免疫三次;每次间隔两周;二免后一周和三免后一周,采用摘除眼球方法收集血液分离抗体,间接ELISA方法测定抗体水平,结果显示:三种重组质粒均能诱导小鼠产生抗PCV-2和PPV的抗体,第三次免疫抗体水平较高,为研制抗PCV-2和PPV核酸疫苗提供了科学依据。
体外表达试验将重组质粒通过Lipofect Transfection Reagent试剂盒转染PK-15细胞,转染48h后收获细胞,运用Trizol法提取细胞总RNA,RT-PCR检测目的基因的转录情况;转染细胞筛选2周后,收获阳性克隆,Western-blotting检测体外蛋白表达情况。RT-PCR检测结果表明,目的基因整合到了细胞的染色体上,并且能够转录到RNA水平;Western-blotting检测结果表明,目的基因能够在体外进行表达,NC膜上得到了Cap蛋白和VP2结构蛋白两条特异性条带,大小分别为28kDa和64kDa,与预期的结果相符,为激发猪对圆环病毒与猪细小病毒进行体液免疫及细胞免疫的多价抗原的研制奠定基础。
Porcine Circovirus Type2 is the primary causative agent of Post-weaning multisys- temic wasting syndrome,Sow abortion and mortality syndrome,Porcine dermatitis and nephropathy syndrome,and so on.PCV-2 could produce lesion of immune function.Because the imm- une function work irregular and prostration, the body could be infect with so much bacteri- um and virus,causing a potential economical impact on the swine industry world- wide.Porcine Parvovirus is the main pathogeny of Sow abortion and mortality syndrome.It possible cause abortion , stillbirth and mummification.Today, PCV-2 and PPV coinfection all over the world,we must to be faced with the draconic challenge.
The PCV-2 and PPV were treated and inoculated into PK-15 cell.The DNA encoding full-length PCV-2 and PPV were extracted using prolease-K. Two pairs of PCR primers were designed according to the sequence of the PCV-2 and PPV 7909 strain.The DNA encoding ORF2 and VP2 were cloned into pCDNA3.1(+) to construct three recombinant plasmids containing ORF2 gene,VP2 gene, and ORF2-VP2 gene, were called pCDNA3.1- ORF2、pCDNA3.1-VP2 and pCDNA3.1-ORF2-VP2. Three recombinant plasmids were further analyzed with restriction enzyme. The result is, objective fragments were inserted into the pCDNA3.1(+) correctly.
We extracted so many recombinant plasmids by the alkaline lysis and polyethylene glycol precipitation method.Analyze concentration and purity coefficient by detecting the Optical Density of the recombinant plasmids. The consequence is that they were good and do not have protein pollution.Healthy mouse were immunized with three recombinant DNA after pre-disposal treatment with Procaine. Injected 0.2mg recombinant DNA by intr- amuscular, two weeks later IM again till three times. We collect the blood-serum through removaling their globes at secondary immunization and third time week-later. Then we established indireet ELISA method for detectting the assays, the result shows that the mouse produce the antibidies to PCV-2 and PPV through IM three recombinant plasmids. Compared the secondary immunization with third times, latter had the conspicuous higher antibody level.
In vitro study, cells that were transfected with pCDNA3.1-ORF2、pCDNA3.1-VP2 and pCDNA3.1- ORF2-VP2 were harvested at 48h postinfection.We extract their genomic RNA to amplified the objective DNA of PCV-2 and PPV with the RT-PCR methods. Cells that were transfected with three recombinant plasmids were harvested at two weeks postinfection. Using the mouse anti-ORF2 and VP2 antibody, the proteins were detected by western-blotting.These results show that ORF2 and VP2 could transcription and translation in transfected PK-15 cells. It is considered that the viral protein of PCV-2 and PPV have the immunogenicity that could stimulatedue the bodies produce the antibody to counteract the disease cause of PCV-2 and PPV. Maybe we could use the expression polyvalent antigen in controlling disease through both humoral-mediated immunity and cell-mediated immunity in the future.
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