【作者】 谭兵；

【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】 粒细胞-巨噬细胞集落刺激因子(granulocyte macrophage colony stimulatingfactor,GM-CSF)是一个具有多项潜能的造血生长因子,不仅能刺激造血祖细胞增殖分化和成熟,而且还能活化树突状细胞及其他单核细胞,在免疫反应中具有重要作用,许多研究证实GM-CSF与DNA疫苗联合免疫,能有效增强DNA疫苗所诱导的免疫应答。马立克病毒(Marek’s disease virus,MDV)的VP22蛋白具有独特的蛋白转导特性,能通过非经典途径从原始表达细胞转导到邻近的非表达细胞中,而且VP22还能使与之融合的外源蛋白在细胞间转导,已被证实具有免疫增强作用。本试验成功克隆了四川山地乌骨鸡GM-CSF基因和MDV的VP22基因,研究了它们对IBV核酸疫苗的佐剂效应,具有重要的学术价值和应用前景,主要研究包括:1.根据GenBank上注册的GM-CSF基因(登录号:AJ621740)序列设计一对引物,采用RT-PCR方法从ConA刺激的四川山地乌骨鸡外周血淋巴细胞总RNA中扩增出鸡GM-CSF基因,克隆入pGEM-T载体,进行序列测定与分析。结果表明,克隆的四川山地乌骨鸡GM-CSF基因全长为435bp,编码144个氨基酸残基。序列分析发现四川山地乌骨鸡GM-CSF(chGM-CSF)基因与其他品种鸡GM-CSF基因核苷酸同源性为99%左右,氨基酸同源率为98%左右,与哺乳动物GM-CSF基因的氨基酸同源性低于30%。蛋白二级预测发现山地乌骨鸡主要含4个α-螺旋,α-螺旋总含量高达55.2%,多处无规卷曲(coil),含量高达36.5%,含有2个β-折叠(strand),含量仅为8.3%,与哺乳动物的GM-CSF二级结构相似,也具有4个α-螺旋,2个β-折叠。四川山地乌骨鸡GM-CSF克隆及序列分析未见相关报道。2.通过PCR方法扩增出不含信号肽的四川山地乌骨鸡GM-CSF成熟蛋白基因,克隆至原核表达载体pET-32a(+),经酶切和测序鉴定正确后,转化入BL21大肠杆菌,经IPTG诱导,SDS-PAGE电泳结果显示融合重组chGM-CSF蛋白主要以包涵体的形式表达,大小约为34 kD左右。通过镍离子金属螯合柱(Ni-NTA)进行重组蛋白纯化,采用纯化后重组蛋白免疫新西兰兔制备兔抗鸡GM-CSF蛋白的多克隆抗体。ELISA结果显示,制备的抗体效价达到了1:3200,Western Blot表明,制备的多克隆抗体能特异性与鸡GM-CSF重组蛋白结合,具有良好的反应原性。本研究获得的chGM-CSF重组蛋白及其多克隆抗体,为研究鸡GM-CSF蛋白生物学功能等提供关键的材料,国内外未见四川山地乌骨鸡GM-CSF基因的原核表达及抗体制备的相关报道。3.通过酶切、连接等方法,将四川山地乌骨鸡GM-CSF基因亚克隆到真核表达载体pVAX1上以构建质粒pVAX1-chGM-CSF,利用菌落PCR和酶切对重组质粒进行鉴定,采用脂质体法转染哺乳动物COS-7细胞,采用间接免疫荧光试验(IFA)和RT-PCR检测质粒在体外的表达情况,收集质粒转染70 h后上清中表达蛋白,通过骨髓细胞增殖试验检测表达的chGM-CSF蛋白的生物学活性。RT-PCR结果显示转染了pVAX1-chGM-CSF质粒的COS-7细胞中能特异性扩增出GM-CSF基因片断,间接免疫荧光分析表明转染了pVAX1-chGM-CSF质粒的COS-7细胞显示出苹果绿荧光,而转染了空质粒pVAX1的细胞则没有显示出任何特异荧光。骨髓细胞增殖试验表明chGM-CSF蛋白能明显促进鸡骨髓细胞的增殖,这些结果为研究chGM-CSF对DNA疫苗佐剂效应提供了关键材料和奠定了理论基础,目前国内未见在COS-7细胞中表达四川山地乌骨鸡GM-CSF及表达蛋白活性分析的相关报道。4.本研究根据发表的马立克病毒VP22的序列(登录号:L10283),设计了一对特异性引物,采用PCR方法从MDV弱毒株的基因组中扩增VP22基因。再根据IBV结构基因S1、M、N序列(登录号:DQ288927),设计引物通过PCR方法扩增出IBV结构基因S1、M、N,分别克隆入真核表达载体pVAX1,酶切鉴定正确后,再通过双酶切,电泳胶回收后与同样酶切处理的VP22片断相连,构建融合表达质粒pVAX-VP22/S1、pVAX-VP22/M、pVAX-VP22/N,酶切和测序鉴定后,通过脂质体转染COS-7细胞,转染40 h后,利用RT-PCR及间接免疫荧光检测重组蛋白在COS-7细胞中的表达。RT-PCR检测结果显示转染了融合表达质粒的COS-7细胞中分别扩增出S1、M、N基因片断,间接免疫荧光分析表明转染了融合表达质粒的COS-7细胞显示苹果绿荧光,而转染了空质粒pVAX1的细胞则没有显示出任何特异荧光。目前国内外未见马立克病毒VP22基因与IBV结构蛋白基因融合表达载体的构建及在COS-7细胞中表达的研究报道。5.为研究chGM-CSF及VP22基因对IBV DNA疫苗的佐剂效应和比较chIL-2、chIL-8、chGM-CSF对DNA疫苗的佐剂效应差异,将大量提取的真核表达质粒,纯化后分别与脂质体溶液混合配制成DNA疫苗,通过腿部肌肉多点注射7日龄雏鸡,21日龄加强免疫一次。在免疫前和免疫后0、7、14、21、28 d采血检测ELISA抗体效价和外周血CD4+、CD8+T淋巴细胞亚群的数量,并在二免后第10d,采用MTT、法检测脾淋巴细胞增殖水平。二免两周后用IBV强毒进行攻毒,观察两周,记录发病及死亡情况,评价疫苗免疫保护力。抗体水平检测结果显示, VP22融合疫苗组pVAX-VP22/S1、pVAX-VP22/M、pVAX-VP22/N产生的抗体水平一直高于对照组pVAX-S1、pVAX-N、pVAX-M,从免疫后第14 d开始,差异显著(P＜0.05),chGM-CSF分子佐剂组pVAX-chGM-CSF+pVAX-S1、pVAX-chGM-CSF+pVAX-N、pVAX-chGM-CSF+pVAX-M抗体水平从免疫后第7 d开始明显高于对照组pVAX-S1、pVAX-N、pVAX-M,且差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组产生的抗体水平高于chIL-2+pVAX-S1、chIL-18+pVAX-S1疫苗组,从免疫后第7 d开始差异显著(P＜0.05);脾淋巴细胞增殖实验显示,VP22融合疫苗组和chGM-CSF分子佐剂组都能有效刺激脾淋巴细胞的增殖,增殖指数高于对照组,且差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组淋巴细胞增殖指数高于chIL-2+pVAX-S1、chIL-18+pVAX-S1组,且差异显著(P＜0.05),其中pVAX-chGM-CSF+pVAX-S1组增殖指数最高,平均值达2.83;外周血CD4+、CD8+T淋巴细胞亚群数量检测结果显示,VP22融合疫苗组淋巴细胞亚群数量免疫后高于对照组,在免疫后21 d开始,差异显著(P＜0.05),chGM-CSF分子佐剂组在免疫后明显高于对照组,从免疫后第7 d开始差异显著(P＜0.05),chGM-CSF+pVAX-S1疫苗组免疫后明显高于chIL-2+pVAX-S1、chIL-18+pVAX-S1组,从免疫后第7d开始差异显著(P＜0.05);攻毒结果表明,chGM-CSF分子佐剂组保护率介于80～90%,高于VP22融合表达的疫苗组的75～80%和对照组65～75%,其中pVAX-chGM-CSF+pVAX-S1为所有组最高,达到了90%,也高于chIL-2+pVAX-S1、chIL-18+pVAX-S1的85%和80%。这些结果表明,chGM-CSF、VP22作为分子免疫佐剂能提高IBV DNA疫苗体液和细胞免疫水平,提高疫苗的攻毒保护率,chGM-CSF对IBVDNA疫苗免疫增强效应要略优于chIL-2、chIL-18。本研究开展的四川山地乌骨鸡GM-CSF和MDV的VP22基因的克隆、表达以及对IBV核酸疫苗佐剂效应的研究,为进一步研制新型、高效、安全禽类DNA疫苗的分子佐剂提供理论依据。更多还原

【Abstract】 Granulocyte-macrophage colony-stimulating factor （GM-CSF） is a glycoprotein that regulates proliferation and differentiation of hematopoietic progenitors. It has a potent effect on differentiation and maturation of dendritic cell （DC）as well as the expression of MHC and co-stimulatory molecules . Co-injection with plasmid DNA expressing GM-CSF has been reported to increase protective immunity induced by plasmid DNA.VP22, a tegument protein that has demonstrated the property of intercellular transport, is capable of distributing fused protein to many surrounding cells. It has been confirmed that it has adjuvant effect on vaccine.In this study, chicken GM-CSF and herpervirus VP22 were cloned and expressed and further used to explore their adjuvant efects on on DNA vaccine （IBV DNA vaccine）.The main content is as follows:1.In this study, according to the gene sequence published in the GenBank （GenBank No: AJ621740）, one pair of specific primer was designed. The Sichuan silky chicken GM-CSF cDNA was isolated from a concanavalin A （ConA）-stimulated peripheral blood lymphocytes using the reverse transcriptase-polymerase chain reaction （RT-PCR）.The GM-CSF cDNA was ligated into the T vector and send to Sequence. Sequence analysis showed that the full-length cDNA is compose of 435 bp ,encoding an open reading frame of 144 amino acids with a predicted molecular mass of 16.15 kD . The deduced amino acid sequence of silky c hicken GM-CSF shares higher 98% and 15%-30% similarity to known GM-CSF in chicken species and other mammalian, respectively. The protein structure prediction showed that the GM-CSF protein has four a- helix and twoβ-strand and much coil. The percentage of a-helix andβ-strand and coil is 55.2%, 8.3%,36.5%,respectively. The c hGM-CSF structure is similar to that of mammalian GM-CSF.2.The GM-CSF without the signal peptide was amplified by PCR and ligated into prokaryotic expression plasmid pET-32a（+） to construct plasmid p32GM-CSF.After identification with restriction enzyme digestion and DNA sequencing,the p32GM-CSF was transformed into BL21 E.coil.The recombinant chGM-CSF was produced with induction by IPTG at 37℃.SDS-PAGE demonstrated that the fusion protein expressed in form of inclusion body is approximately 34 kD in size. The acquired recombinant protein was purified by Nickel ion affinity chromatography and used to immunize rabbbit to prepare the Polyclonal antibodies （pAbs） specific for chicken GM-CSF .The ELISA assays showed that the antibody titre is very high and reach 1:3200. Western blotting analysis showed that it has good reactionogenicity and could bind with recombinant chGM-CSF protein specially expressed in E.coil.The acquired chGM-CSF protein and specific Polyclonal antibodies would offer critical material to the furt her research on biological activity of chGM-CSF.3.The full-length chGM-CSF,which was obtained from the previous constructed plasmid pGM-CSF, was ligated into the plasmid pVAX1.After identification by restriction enzyme digestion and DNA sequencing,the plasmid pVAX1-chGM-CSF was transfected into COS-7 cell by lipofectamine. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The recombinant chGM-CSF protein was prepared from COS-7 cell expressing supernatant 70 h after transfecting.The biological activity of chGM-CSF was measured by bone marrow cell proliferation assay. The results of restriction enzyme digestion and DNA sequencing showed that the plasmid pVAX1-chGM-CSF were constructed successfully. The mRNA transcription of chGM-CSF gene could be detected by RT-PCR in the COS-7 cells which were transfected with plasmids pVAX1-chGM-CSF. The expression of chGM-CSF protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with plasmids.The bone marrow cell proliferation assay showed that chGM-CSF protein could enhanced bone marrow cell proliferation obviously. This study would provide materials for the further development of DNA vaccine against IBV.4.According to VP22 gene sequence（GenBank No:L10283 ） already published in t he GenBank, one pair of specific primer was designed.T he VP22 gene was amplified from the MDV genome by PCR.Then, According to the sequence of IBV S1,M,N gene, the specific primer of S1,M,N gene were designed,respectively.The S1,M,N gene were amplified by PCR and ligated into the plasmid pVAX1 to construct recombinant plasmid ,respectively.After identification by restriction enzyme digestion and DNA sequencing,these recombinant plasmid were digested by two restriction enzyme,respectively and the big fragment of each recombinant plasmid was purified by agar electrophoresis. To construct fusion plasmid pVAX-VP2/S1 ,pVAX-VP22/M,pVAX -VP22/N,the each big fragment ligated with VP22 fragment dealed with same restriction enzyme,respectively.After identification by restriction enzyme digestion and DNA sequencing,the fusion plasmid pVAX-VP22/S1. pVAX-VP22/M, pVAX-VP22/N was transfected into COS-7 cell by lipofectamine,respectively. The expression of heterologous genes were detected by RT-PCR and indirect immunofluorescence assay. The results of restriction enzyme digestion and DNA sequencing showed that these fusion plasmid were constructed successfully. The mRNA transcription of S1,M,N gene could be detected respectively by RT-PCR in t he COS-7 cells which were transfected with these fusion plasmids. The expression of S1,M,N protein could be detected by indirect immunofluorescent assay in the COS-7 cells which were transfected with these fusion plasmids.This study would provide materials and foundation for the further evaluating the adjuvant effection of VP22 for IBV.DNA vaccine.5.In order to evaluate the immune-modulatory of VP22 and GM-CSF as a genetic adjuvant, the plasmids used for vaccination were extracted from E.coil and purified by a C hinese invention patent method which was established previously in our lab. The diluted plasmids were encapsulated by liposome in equal volume, and administered to the 7-day-old chickens by intramuscularly injection. After two weeks later, the chickens were boosted by DNA vaccine. Indirect ELISA methods and the Fluorescence activated cell sorter were used to detect anti-IBV antibody and the number of CD3+, CD4+and CD8+ T lymp hoctyes on day 0, 7,14,21,28 after immunization. Ten days after boosting, MTT assay were used to detect the proliferation level of immunized chicken.Two weeks after boosting, all chicken were challenged by virulent IBV strain and observed for two week. The results of specific antibody show that there was a significant difference （P＜0.05） in ELISA antibody levels between VP22 fusion group pVAX-VP22/S1 ,pVAX-VP22/M, pVAX-VP22/N and control group p VAX-S1、pVAX-N、pVAX-M since the 14t h-day after first inoculation. There was also a significant difference （P＜0.05 ） in ELISA antibody levels between chGM-CSF as molecular adjuvant group pVAX-chGM-CSF+pVAX-S1,pVAX-chGM-CSF+pVAX-N, pVAX-chGM-CSF+pVAX -M and control group since the 7th-day after first inoculation.The antibody level of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1.The results of lymphocyte proliferation showed that there was a significant difference （P＜0.05） between VP22 fusion group and control group.There was also a significant difference （P＜0.05）between chGM-CSF as molecular adjuvant group and contol group. The SI index of lymphocyte proliferation of pVAX-chGM-CSF+pVAX-S1 is 2.83,significant higher than those of pVAX-chIL-2+pVAX-S1,pVAX-chIL-18 +pVAX-S1.The percentage of CD4+, CD8+ T-lymphocytes from chickens immunized with VP22 fusion group was significantly higher （P＜0.05） than t hat of control group since the 21d after the first vaccination. W hen compared to that of control group,the significantly higher percentage of CD4+, CD8+ T-lymphocytes was observed in group immunized with chGM-CSF as molecular adjuvant since the 7^th day after the first vaccination（P＜0.05）.The percentage of CD4+, CD8+ T-lymphocytes of pVAX-chGM-CSF+pVAX-S1 was significant higher than pVAX-chIL-2+p VAX-S1, pVAX-chIL8+p VAX-S1. The result of virus-challenge assay showed that the protection rate of chGM-CSF as molecular adjuvant group was from 80% to 90%, higher than 75～80% of VP22 fusion group and 65～75% of control group.In all group, protection rate of the group immunized with pVAX-chGM-CSF+pVAX-S1 is highest and reach 90%,higher than 85%, 75% of pVAX-chIL-2+pVAX-S1, pVAX-chIL-18+pVAX-S1, respectively, far from 80% induced by inactivited vaccine. These results suggested that VP22 and chGM-CSF as a genetic adjuvant is feasible to enhance the immune response of IBV DNA vaccine .The chGM-CSF as molecular adjuvant is better than IL-2,IL-18 in the immune-modulatory of IBV DNA vaccine .These results provide a new way to develop effective avain vaccine.更多还原