【作者】 李殿明；

【导师】 包振民；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 猪瘟(Hog Cholera, HC)是由猪瘟病毒(Classical swine fever virus,CSFV;又称Hog cholera virus,HCV)引起的猪的急性高度接触性传染病,对养猪业危害极大。虽然随着许多综合防治措施的实施,已控制了猪瘟的大规模流行,但该病仍在我国广大养猪地区不断发生和小规模流行,每年均使养猪业蒙受巨大的经济损失。我国每年有3%的饲养猪死于猪瘟,其中90%是仔猪。并且近年来,该病在美洲、亚洲、欧洲等国家和地区呈现广泛流行的趋势,一些宣布已消灭了猪瘟的国家如法国、荷兰、德国、比利时等又见猪瘟复发的报道。研制更为有效的猪瘟疫苗和治疗药物已成为养猪业的迫切需要。E2(gp55)蛋白是CSFV病毒主要的抗原蛋白,也是三个病毒糖蛋白(E0、E1、E2)中保守性最低,最易变异的分子。实验证明,E2蛋白在体内可诱导产生病毒的中和抗体并激发对CSFV的免疫保护,使猪能够抵抗致死量CSFV的攻击。E2蛋白作为CSFV囊膜糖蛋白中最重要的一个保护性抗原蛋白,一直是国内外学者的主要研究对象。本研究结合LTB的粘膜免疫佐剂特性,选择了猪瘟病毒囊膜糖蛋白E2的部分保守抗原决定簇以及公认的有效粘膜免疫佐剂——大肠杆菌肠毒素B亚单位(LTB)的基因作为研究对象,参考毕赤酵母的密码子偏好性,人工合成了编码E2蛋白保守抗原决定簇和编码LTB的融合蛋白(E2-LTB)的编码基因。将该基因克隆到毕赤酵母表达载体ppicZαa上,构建了重组表达载体ppicZaAE2-LTB,通过电转化使ppicZaA E2-LTB稳定整合到了P. pastoris宿主菌GS115中,成功构建了分泌表达工程菌ppicZaA E2-LTB/ GS115,诱导表达实验结果表明,目的蛋白E2-LTB能被毕赤酵母分泌到细胞外,通过western-blot分析表明该表达产物能与CSFV抗血清以及兔抗LTB血清反应,融合蛋白能与神经节苷脂受体结合,说明该融合蛋白具有良好的抗原性和生物学活性。在此基础上我们进一步对所构建融合蛋白酵母表达工程菌pPICαA-E2-LTB/ GS115的发酵参数进行了优化,以摇瓶培养方式探讨不同pH值,不同甲醇浓度,不同诱导时机以及不同诱导时间对外源蛋白表达的影响;以NBS BIOFLO 4500型30L发酵罐进行高密度发酵条件探索;利用硫酸铵分级分离、液相色谱对目的蛋白进行纯化。结果表明在pH=6.0的条件下以1%甲醇诱导表达96小时后,目的蛋白E2-LTB表达最优;在高密度发酵条件下,目的蛋白的理论表达量可以达到约1g/L,最终收率能够达到400mg/L发酵液,蛋白最终纯度达到95%以上。利用pPICαA-E2-LTB/ GS115工程菌分泌表达并经过纯化的E2-LTB融合蛋白作为一种粘膜免疫疫苗,采用口服和鼻饲两种方式对小鼠进行接种免疫实验,通过ELISA检测抗体水平、细胞因子水平对免疫学效力进行评估。结果表明:鼻饲、口服免疫途径都能激发产生IgA和IgG抗体,其中鼻饲产生的抗体效价高于口服免疫。使用融合蛋白产生的抗体明显高于单独使用E2蛋白组;无论是在免疫小鼠的血清中,还是在体外培养的小鼠脾细胞中,鼻饲E2-LTB都能增强IL-2、IL-5和INF-γ的分泌。对E2-LTB免疫3次的家兔接种100MID50剂量猪瘟兔化弱毒(HCLV)进行的免疫攻毒保护试验表明,空白对照组无保护作用,E2蛋白免疫组具有一定的免疫保护作用,而E2-LTB融合蛋白免疫组则完全能够抵抗猪瘟病毒的攻击。说明该黏膜免疫疫苗对实验动物具有良好的病毒保护能力。以纯化的E2蛋白为抗原包被酶标板,通过对ELISA各反应条件的优化,确定了最佳反应条件:重组E2蛋白的最佳包被浓度为2.5ug/ml,最适包被条件为4℃24h,封闭液为加入0.02%硫柳汞、2mMEDTA、1%BSA,PBS,最佳封闭条件为37℃1h;最佳血清稀释倍数为100倍,酶标兔抗猪IgG最适工作浓度为1:1200,用含有4%PEG 6000的稀释液稀释二抗,其最佳工作时间为20min。通过试剂盒的特异性和敏感性实验以及重复实验表明该试剂盒具有特异性好,敏感性高,稳定性强等特点,其阴阳性值临界点OD值为0.15,对60份检测样品检验结果表明与进口试剂盒符合率达到83.3%。更多还原

【Abstract】 Hog Cholera (HC) is an acute and highly contagious disease that has been a great threat to the global swine industry. This disease is caused by classical swine fever virus (CSFV), which is also named as Hog cholera virus (HCV) infection. Large-scale epidemic spread of HC has been controlled due to various comprehensive measures taken in recent years. However, occasional sporadic HC prevalence occurs constantly in our country and results in significant economical lost. Statistical data indicates that annually 3% pigs, 90% of them are young pigs, are dead of HC in China. To make it even worse, extensive HC spreading tendency has been detected in some America, Asia, Europe countries or regions. HC cases were reported in even some countries such as France, Netherland, Germany and Belgium that had declared extinction of HC. Glycoprotein E2 of CSFV is the primary antigenic protein with high variability, which can induce virus-neutralizing antibodies against CSFV and resisted lethal CSFV challenge. This protein has been studied widely all the word as an important protective antigenic protein.The DNA sequence encoding swine fever disease virus (CSFV) glycoprotein E2 antigenic epitope and Escherichia coli Heat-labile Enterotoxin B subuint (LTB) gene with codons preferred by the methylotropic yeast pichia pastoris were synthesized and inserted into pPICZaA expression vector. After being linearized by digestion, the vector with AOX1 promoter and a -factor secretion signal sequence was transformated into Pichia pastoris (GS115) by electroporation to integrate with the genome. The transformants with high copies were screened by Zeocinn? and were induced with methonl to express the recombinant protein. SDS-PAGE and Western blot analysis showed that the fusion protein successfully secreted into the culture medium and retained the antigenicity associated with LTB and CSFV antibodies.The optimized expression conditions of the acquired positive recombinant yeast strains P. pastoris GS115- pPICαA-E2-LTB was established by studying the relations between expression yield and growth conditions with different induction time, strain density, pH value and dose of methanol, respectively. And the conditions that the strain zymolysised in fermenter (NBS BIOFLO 4500) with higher density was optimized, and then the recombinant protein was purified by stepwise precipitation with ammonium sulfate and HPLC. The optimal conditions of the recombinant protein expression were planted in medium with 30℃, pH 6.0 and incubated 96h with 1.0% methanol. After induction under optimal conditions with high density,the maximum yield of recombiant protein could reach to 1g/L theoretically. In this study, yield of E2-LTB protein was 400mg/L, and the concentration of E2-LTB was 95%. It indicated that the strain was character of good stability, higher output and adapt to product cosmically.A positive recombinant strain Pichia pastoris, expressing swine fever virus (CSFV) glycoprotein protein E2 epitopes and E. coil heat-labile enterotoxin B Subunit (LTB), was used to produces mucosal vaccine E2-LTB. The antibody and cytokines response to E2-LTB vaccine were evaluated by ELISA. Remarkably higher levels of antiviral IgA and IgG antibodies were induced in serum, nasal wash and lung wash by routes of intranasal and perorall vaccination, respectively. All antibodies induced by intranasal rout were higher than that induced by perorall rout. E2-LTB could directly enhance the expression of T-cell-mediated interleukin-2 (IL-2), IL-5 and IFN-γin both the serum of mouse and the cultured mouse spleen cells. In virus challenge experiments, all the rabbits were challenged with 100MID50 HCLV. E2 Eimmunized Rabbits showed a mild increase of body temperature, while no E2-LTB Eimmunized rabbit increased its body temperature. On the other hand, the body temperature of control rabbits increased, but then recovered normally. These results demonstrated that the E2-LTB could induce an efficient immune protection against HCLV infection, and might provide a new kind of vaccine for CSFV.An indirect ELISA Kit was constructed to detect antibody against CSFV by coating the wells of 96-well plate with purifted E2 protein.Various factors and conditions of ELISA were explored,and the optimal reaction conditions of ELISA were determined.The optimal concentration of recombinant E2 protein for plate coating was 2.5μg/mL,and the optimal coating condition of recombinant E2 protein for ELISA was at 4℃for 24h. The blocking agents were l% BSA, 0.02% thimerosal and 2mMEDTA, and the blocking time was 1 hours at 37℃. The dilution of serum sample was l:100,and the dilution of HRP-labeled rabbit anti-porcine lgG was l:1200. The samples for ELISA were incubated at 37℃for 20min before terminated with the stopping solution.The indirect ELISA Kit by the purified recombinant nucleoprotein had good specificity for the detection of CSFV antibody in serum.The difference value among wells in a plate and among plates for ELISA was both less than 6%,which showed the assay had a good retrievality.60 serum samples from swines were detected for the antibody to CSFV by using our developed ELISA and the IDEXX CSFV Antibody Test Kits simultaneously. It was found that our developed ELISA had the highly sensitivity and correspondence, and the Cut off value of the kit is 0.15 between positive serum and negative serum. The correspondence between the ELISA kit and the IDEXX kit was 83.3%更多还原