【作者】 张建远；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2009， 博士

【摘要】 猪圆环病毒病由猪圆环病毒2型(Porcine circovirus type2,PCV2)所致,该病除表现为猪断乳后多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)外,还与猪皮炎肾病综合征(porcine dermatitis and nephropathy syndrome,PDNS)、猪呼吸道疾病综合征(Porcine respiratory disease complex,PRDC)、增生性坏死性肺炎(Proliferative and necrotizing pneumonia,PNP)、先天性震颤((CongenitaltemorAll,CT-All)等猪圆环病毒相关疾病(PCVAD)有关。猪圆环病毒病在世界不同的国家与地区引致广泛流行与致病,也是我国规模式化猪场主要传染病之一,给世界养猪业造成巨大的经济损失,引起全世界高度关注。目前,国内还没有猪圆环病毒2型商品化疫苗,国外的疫苗研究也在近年来才开展。研究新型猪圆环病毒2型核酸疫苗提高疫苗免疫的效果,对于防制猪圆环病毒病有重要意义。本研究分离鉴定了猪圆环病毒2型病毒株,构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚糖复合物,开展猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价筛选猪细胞因子分子免疫佐剂提供理论依据和实验数据。主要研究结果如下:1.猪圆环病毒2型病的检测及病毒株的分离鉴定。采用ELISA技术对9个规模化猪场987头猪的血清进行了猪圆环病毒2型抗体检测,结果表明:受检未免疫猪血清中,圆环病毒2型抗体阳性率为74.3%;采用PCR检测了可疑病料104头,可疑病料中PCR检测阳性率为77.9%,从病料中分离鉴定了一株猪圆环病毒2型(PCV2whn),在透射电镜下观察到持续感染猪圆环病2型的PK15细胞胞浆及细胞核内有包涵体,以及呈晶格状排列的病毒粒子,直径18±2nm;测定了PCV2whn分离株的全基因组序列,猪圆环病毒2型分离株的基因组全长为1767bp,与我国不同地方的猪圆环病毒2型病毒的分离株基因组的同源性高达92.7-97.5%,与欧洲、美洲、南非分离株的同源率在94.2-96%之间。本研究自主分离鉴定获得新的猪圆环病毒2型分离株,为猪圆环病毒2型研究获取了新的材料。2.pVAX1-ORF2-ISS核酸疫苗构建及核酸疫苗壳聚糖纳米颗粒制备。选用pVAX1为真核表达载体,以猪圆环病毒2型ORF2为构建目的基因,合成插入含11个CpG motif全长92bp的ISS序列,采用PCR扩增PCV20RF2片段,通过酶切及连接等操作将两段序列亚克隆到pVAX1载体中,构建了pVAX1-ORF2-ISS核酸疫苗,并采用酶切鉴定所构建的真核表达载体。结果显示两个片段正确插入到载体中。使用壳聚糖对pVAX1-ORF2-ISS核酸包被,通过透射电镜和胶阻滞分析,结果显示,pVAX1-ORF2-ISS与壳聚糖结合,成功构建与制备pVAX1-ORF2-ISS核酸疫苗壳聚糖复合物。本研究以pVAX1为优化载体,合成插入ISS序列,设计构建了含ISS与ORF2的pVAX1-ORF2-ISS新型核酸疫苗,此研究尚未见报道。3.猪白细胞介素IL-4、IL-6、IL-6/4的鉴定及其分子佐剂壳聚糖纳米颗粒的制备。选用“四川大学“动物疫病防控与食品安全四川省重点实验室”提供的猪白细胞介素IL-4、IL-6、IL-6/4真核表达质粒,利用酶切方法对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了鉴定,结果证明3种白细胞介素的目的片段大小与预期大小符合。使用壳聚糖对猪白细胞介素IL-4、IL-6、IL-6/4的真核表达载体进行了包被,通过透射电镜和胶阻滞分析结果显示,真核表达质粒全部与壳聚糖结合,成功制备猪白细胞介素IL-4、IL-6、IL-6/4的分子免疫佐剂壳聚糖纳米颗粒。本研究应用成功制备的猪白细胞介素IL-4、IL-6、IL-6/4真核表达分子免疫佐剂质粒与猪圆环病毒2型核酸疫苗进行的联合免疫实验,在国内外未见报道。4.猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子联合免疫猪。使用构建和制备的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫实验猪,通过测定不同分组仔猪免疫后的特异性抗体滴度以及T淋巴细胞亚类CD3+、CD4+、CD8+细胞免疫变化情况,对pVAX1-ORF2-ISS核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫效果作出了分析和评定。同时,使用pVAX1-ORF2-ISS核酸疫苗在小白鼠体上做安全性实验。结果显示,pVAX1-ORF2-ISS核酸疫苗能够诱导仔猪产生特异性抗体和T淋巴细胞亚类的免疫应答,免疫期持续60d以上,能诱导细胞免疫并产生特异性抗体,pVAX1-ORF2-ISS核酸疫苗与本课题组构建保存的pVAX1-ORF2比较,其免疫效果优于pVAX1-ORF2(P＜0.05),与空载体及空白对照组相比,差异极显著(P＜0.01);3种猪细胞因子分子佐剂与pVAX1--ORF2-ISS核酸疫苗同时免疫仔猪能显著提高pVAX1-ORF2-ISS核酸疫苗的免疫效果(P＜0.05),以IL-6、IL-6/4为佳,IL-4次之;核酸疫苗的安全性试验表明pVAX1-ORF2-ISS核酸疫苗安全。同类研究均以小鼠进行免疫实验,而本研究用猪圆环病毒2型新型核酸疫苗免疫及与猪细胞因子分子佐剂联合免疫猪未见报道。本研究用构建pVAX1-ORF2-ISS核酸疫苗和制备核酸疫苗壳聚纳米颗粒,开展的猪圆环病毒pVAX1-ORF2-ISS核酸疫苗及与猪细胞因子佐剂联合免疫猪研究,为研制新型、高效、安全的猪圆环病毒2型核酸疫苗及测定评价猪细胞因子分子免疫佐剂提供了理论依据和实验数据。更多还原

【Abstract】 Porcine circovirus virus（PCV2） is recognized as the essential infectious agent of postweaning multi-systemic wasting syndrome（PMWS）,porcine dermatitis and nephropathy syndrome（PDNS）,porcine respiratory disease complex（PRDC）, proliferative and necrotizing pneumonia（PNP） and congenital temorall（CT-All）.It is epidemic in many countries in the world and is also a major infectious disease in intensive pig farms of China.It caused a great economic loss to world’s pig industry. Therefore,there are many scientists and other people engaging pig raising paying much more attention to PCV2.However,there has not effective commercial vaccine in China and only a few researches for PCV2 vaccine in foreign countries up to now.Thus,it is very important to develop novel PCV2 DNA vaccine and apply cytokine molecular adjuvant to improve immune effects of vaccine.This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.The aim of this study was to improve the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for prevention of PCV2.The main content of the research is as follows.1.The isolation and identification of PCV2 Sichuan strain.Using ELISA to detected blood serum antibody of 987 pigs from 9 farms revealed that the PCV2 positive rate was 74.3%.104 specimen of dead pigs from 9 pig farms were identified as PCV2 by PCR and the virus were isolated.The results showed that the PCV2 positive rate of the specimen was 77.9%;A PCV2 whn strain was isolated,presented inclusion body in cell plasm and cell nuclear of PK15 cell,and the virion whose diameter was 12±2nm, presented crystal lattice arrangement by electron microscope;The complete genome of the virus was sequenced and the full-length was 1767nt,which showed 92.7-97.5% genetic homology with the isolates of China and 94.2-96%with European,American and South Africa isolates.We are successfully isolated and identificatied PCV2 in Sichuan province and got accession number in Genbank,which offered new materials to research of PCV2.2.The construction of pVAXI-ORF2-ISS DNA vaccine and the preparation of the DNA vaccine-chitosan-nanoparticle（CNP） complex.The pVAX1-ORF2-ISS DNA vaccine was constructed through the following steps.ORF2 of PCV2 and 11 CpG motif as ISS sequence were inserted into pVX1 eukaryotic expression vector.Then identified by enzyme digestion analysis.The results showed that the two fragments were rightly inserted into vector.CNP was used to coating pVAX1-ORF2-ISS plasmid.The transmission electron microscope and glue blocking analysis indicated that vaccine plasmids combined with CNP and pVAX1-ORF2-ISS DNA vaccine were successfully constructed and prepared.We had successfully constructed a kind of pVAX1-ORF2-ISS novel DNA vaccine with pVAX1 vector and ISS,which was reported at the first time.3.The identification of porcine cytokine molecular IL-4,IL-6 and IL-6/4 and the preparation of CNP as molecular adjuvant.The porcine cytokine molecular IL-4,IL-6 and IL-6/4 eukaryotic expression plasmid offered by ministry of education key laboratory for Sichuan university biological resource and ecological environment.Enzyme digestion analysis was applied to identify the three kinds of plasmid and the results showed that the length of target fragments accorded with expectations.CNP was used to coating IL-4, IL-6 and IL-6/4 plasmid.The transmission electron microscope and glue blocking analysis indicated that all the plasmids combined with CNP and cytokine molecular adjuvant were successfully prepared.We successfully prepared IL-4,IL-6 and IL-6/4 eukaryotic expression vector,which was reported at the first time.4.The combined immunization experiment on pigs using PCV2 pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.By testing piglets in every experimental group of specific antibody titers and the quantity of lymphocytes CD³⁺, CD⁴⁺ and CD⁸⁺,analysis and evaluation were achieved about the pVAX1-ORF2-ISS DNA vaccine and porcine cytokine molecular adjuvant.Moreover,safety experiment was performed using white mice.The results showed that pVAX1-ORF2-ISS DNA vaccine could induce the generation of specific antibody and cellular immunity responses,and the duration of immunity could last over 60 days.The effects of pVAX1-ORF2-ISS and pVAX1-ORF2 DNA vaccines were also compared.The result proved that pVAX1-ORF2-ISS was better than pVAX1-ORF2 DNA vaccine（P＜0.05） and the difference was extremely significant（P＜0.01） when compared with empty vector and blank control groups.Also,the three cytokine molecular adjuvant could extremely significantly improve the immune effects of pVAX1-ORF2-ISS DNA vaccine（P＜0.05）. IL-6 was the best,followed by IL-6/4 and IL-4.DNA vaccine safety experiment indicated that the pVAX1-ORF2-ISS DNA vaceine was safe and reliable.White mice were common used to study PCV2 DNA vaccine of pVAX1-ORF2-ISS and combined immunization with porcine cytokine molecular adjuvant,and it was the first time using pigs as animal model,which could get more practical experimental data.This research was carried out to construct novel PCV2 DNA vaccine,prepared vaccine with CNP complex and did combined immunization with porcine cytokine molecular adjuvants.It had improved the immunity of PCV2 DNA vaccine and evaluate the effects of porcine cytokine molecular adjuvants,which supply theoretical basis and experimental data for the research of novel,effective and safe vaccine of PCV2.更多还原