【作者】 卢亚楠；

【导师】 徐永平；

【作者基本信息】 大连理工大学 ， 生物化工， 2009， 博士

【摘要】 对虾白斑综合症(White Spot Syndrome,WSS)是目前危害对虾养殖业最为严重的病害之一,在世界范围内已造成了巨大经济损失,但目前仍未得到有效的防治。因此,寻找一种安全、简便、高效的防治方法,已成为解决该病害的关键。卵黄免疫球蛋白(Egg yolk immunoglobulin,IgY)可用于治疗由细菌或病毒等多种病原体引起的人类及动物疾病,具有安全无残留、不产生抗药性、方便易得、稳定性高等优点,特别是可以应对复杂的水体环境,针对性地使无脊椎动物获得被动免疫保护,在防治一些水产动物疾病方面,具有广阔的应用前景。本研究对WSSV进行了扩增、分离、鉴定,以其DNA为模板,利用PCR技术分别钓取WSSV的主要致病蛋白基因VP28、VP19、VP15,并设计两条连接肽GGDAGD和GNSPNA,通过重叠延伸PCR技术将三段基因串联构成融合基因。将这些目的基因与真核表达载体pCI连接构建单一基因重组质粒pCI-28、pCI-19、pCI-15以及融合基因重组质粒pCI-28-19-15。通过原核细胞诱导表达和转染HeLa细胞使其表达目的蛋白,运用Western Blotting及间接免疫荧光反应检测DNA疫苗的免疫原性。结果表明,目的基因能够进行表达,并能够特异性的识别WSSV抗体。从而,证明了WSSV的DNA疫苗具有良好的免疫原性,可以表达出目的蛋白并对相应的抗体进行识别,为其作为抗原免疫蛋鸡奠定基础。以WSSV病毒灭活疫苗为对照,将上述制备的DNA疫苗作为抗原,胞嘧啶-鸟嘌呤(CpG)二核苷酸的寡核苷酸链(CpG oligodeoxynucleotides,CpG ODNs)作为DNA疫苗佐剂免疫蛋鸡,获取抗WSSV特异性卵黄抗体。通过间接ELISA法监测抗体活性,结果表明,CpG佐剂能够显著增强DNA疫苗的免疫性能(P＜0.05);以不同的抗原免疫蛋鸡均可获得特异性卵黄抗体,特异性IgY在110天仍保持较高的抗体活性;灭活疫苗组及DNA疫苗组的抗体活性与阴性对照组均有显著差异(P＜0.05)。融合基因DNA疫苗与单一基因DNA疫苗免疫产生的特异性IgY活性之间无显著差异;单一基因DNA疫苗免疫效果与其混合交叉免疫的效果也无显著差异;采用水稀释,两步盐析,超滤法从卵黄中分离纯化IgY,平均每枚蛋黄可以获得120mg以上的IgY,特异性IgY含量随时间变化趋势可以反映出抗体活性的变化。不同疫苗的抗体产物的总IgY含量基本一致;而特异性IgY含量则不同,DNA疫苗获得的抗体产物中特异性IgY含量明显高于对照,低于灭活疫苗。使用制备的IgY蛋白粉和卵黄粉,通过注射、口服、浸泡等方式系统地考察了抗WSSV的特异性卵黄抗体对实验动物被动免疫保护效果。体外中和注射实验结果表明,特异性IgY能够一定程度上抑制WSSV:在口服被动免疫实验中,1%灭活疫苗IgY蛋白粉的保护效果最佳,累计死亡率53.3%,口服10%灭活疫苗卵黄粉的保护效果其次,累计死亡率为66.7%;浸泡被动免疫实验中,灭活疫苗IgY浸泡组的死亡率为33.3%,DNA疫苗IgY浸泡处理组累计死亡率为80%,以上处理均与对照组呈显著性差异(P＜0.05),表明以注射、口服及浸泡方式,将制备的特异性IgY被动免疫克氏鳌虾,具有抑制WSSV的效果。综上所述,本研究制备的抗WSSV特异性IgY具有一定的被动保护作用,其作为一种新型的水产动物抗病毒免疫制剂,将具有良好的产业化前景。更多还原

【Abstract】 White spot syndrome(WSS),is one of the most virulent diseases,causing high mortality and large economic losses in cultured shrimp worldwide.Up to date no effective measuer is available to pervent the spreading of WSSV.Therefore,it is urgent to find an effective method to control this pathogen.Chicken egg yolk immunoglobulin(IgY) provides an inexpensive,effective and stable source of antibodies for the passive immunization of animals suffering from different kinds of diseases.To complicated water environment and invertebrates lack a true adaptive immune response system,the passive immunization of specific IgY is a promising immunity preparation for the treatment and prevention of aquatic diseases.In this study,WSSV were amplified,separated,identified.The major antigen region gene VP28,VP19,VP15 of WSSV were amplified by PCR,and then linked together by overlap extension PCR with the designed linkers(GGDAGD and GNSPNA) as overlap regions.The single gene and fusion gene was cloned into the eukaryotic expression plasmid pCI,and then pCI-28,pCI-19,pCI-15,pCI-28-19-15 DNA vaccine was sucessfuly constructed.The E.coli BL21 cells and HeLa cells were transfected by DNA vaccine and the expression of goal gene was detected by Western Blotting and indirect immunofluorescence assay(IFA).The results showed genes that could express and recognise antibody,and these DNA vaccines had good immunogenicitySubsequently,hens were immunized with recombinant plasmids as antigens which used CpG oligodeoxynucleotides(CpG ODNs) as adjuvant,compared with inactivated WSSV vaccine.And the activity of IgY was detected by indirect enzyme-linked immunosorbent assay(ELISA).The results demonstrated that the CpG ODNs enhanced the immune response of DNA vaccine significantly(P＜0.05).The DNA vaccine and inactivated WSSV showed significant difference compared with negative control(P＜0.05).Anti-WSSV IgY still maintained high antibody activity on day 110.But There was no significant difference between single DNA groups.The single DNA group showed no significant difference compared with fusion gene DNA group and the mixture group.IgY was purified from the water-soluble fraction by a combination of several purification techniques including salt precipitation,ultrafiltration and freeze-drying.The yield of IgY is above 120mg/egg,and the contents of specific IgY in yolk varied at the same trend to that of IgY tite.The average contents of total IgY from different antigens are consistent.But the contents of specific IgY from DNA vaccines are more than negative control,and less than inactivated WSSV.The protective effects of IgY powder and egg yolk powder against WSSV in the experiment ainimals were evaluated.In viral neutralization experiment by intramuscular injection,specific IgY against WSSV could inhibit WSSV to some extent.In oral administration experiment,those fed a diet containing 1%IgY power of inactivated WSSV and 10%egg yolk powder showed mortalities of 53.3%and 66.7%,respectively.The inactivated WSSV immersion group showed a mortality of 33.3%,and the DNA immersion group was 80%.These results have significantly differences compared to the positive control group which showed a mortality of 100%(P＜0.05).The results suggested that passive immunization of specific IgY antibodies through oral administration and immersion were effective to protect crayfish against WSSV.In conclusion,the specific IgY preparation in this study can provide a novel approach to the prevention and treatment of WSSV infection,which will have a industrial prospect.更多还原