重组鹅IL-17对鹅细小病毒VP3黏膜免疫增强作用的研究
摘要
动物传染病原的传播与侵染大多数都通过消化道和呼吸道为主要的侵入途径，因此动物机体的黏膜免疫系统在抵抗致病微生物的入侵中体现出十分重要的作用。对黏膜免疫系统的有效刺激需要满足两个基本条件，一是疫苗中的抗原能够有效地被传递到黏膜淋巴组织，二是安全有效的黏膜佐剂加强免疫应答。免疫佐剂是本身不具备抗原性，掺入疫苗制剂后能促进疫苗抗原特异性免疫应答的物质。近年来的研究发现，新型Th细胞——Th17所分泌的主要效应因子IL17，具有强大的招募激活中性粒细胞，以及促进T细胞反应的作用，是宿主抵抗特殊病原体的重要而独特的细胞因子。
目前相对于哺乳动物黏膜免疫的研究，禽类黏膜免疫的研究相对缓慢，而且大部分研究都集中在鸡的相关研究。本研究首次克隆东北白鹅IL-17、CD4和CD8α基因，以真核表达的东北白鹅IL-17作为黏膜免疫佐剂与鹅细小病毒（GPV）的衣壳蛋白VP3病毒样粒子联合免疫，或东北白鹅IL-17与VP3融合表达后免疫东北白鹅，通过检测体液免疫和细胞免疫的多项指标对免疫效果进行评价，证实东北白鹅IL-17作为黏膜免疫佐剂可以有效诱导机体黏膜免疫和系统免疫，对小鹅瘟的防治及开发有效的黏膜疫苗具有十分积极的意义。
1东北白鹅IL-17、CD4和CD8α基因的克隆及序列分析
首先，从东北白鹅的胸腺组织中提取总RNA，经过SMART反转录酶合成双链cDNA，经PCR分别获得东北白鹅IL-17、CD4和CD8α基因，将扩增基因克隆至克隆载体并测序。
用DNAMAN6.03软件将测序的结果分别在美国国立生物技术信息中心（NCBI）的上进行Blast分析，与数据库中的序列进行相似性的比对。可以确定所克隆的基因为东北白鹅IL-17、CD4、CD8α基因，将序列提交至NCBI并获得登录号。确认克隆正确的东北白鹅IL-17、CD4和CD8α基因翻译成蛋白后用ClustalX2.1分别和NCBI中已经发表的各个物种的蛋白质序列进行蛋白序列同源性分析，然后再应用MEGA5.0生物软件程序以Neighbor-joining算法构建生物进化树。分别应用在线分析软件SignalP4.1Server和TMHMM Server v.2.0对东北白鹅IL-17、CD4和CD8α蛋白进行信号肽、跨膜区的预测分析，以便于进行下一步对成熟的东北白鹅IL-17（mGoIL-17）和东北白鹅CD4、CD8α胞外区的表达。
2mGoIL-17、东北白鹅CD4和CD8α胞外区原核表达及多克隆抗体的制备
根据东北白鹅IL-17的信号肽分析结果以及东北白鹅CD4和CD8α的胞外区的分析结果，设计表达引物扩增得到mGoIL-17、东北白鹅CD4和CD8α的胞外区，连接至pET-32a或pET-30a载体，然后转化至大肠杆菌Rosetta感受态细胞，IPTG诱导表达蛋白。采用Ni-NTAHis柱纯化方法纯化蛋白，免疫新西兰白兔制备多抗血清，通过western blot确定血清的反应性，为后续试验奠定基础。
3mGoIL-17和mGoIL-17融合GPV-VP3的真核表达及活性的测定
将pEASY-Blunt-mGoIL-17质粒通过双酶切得到IL17基因，克隆至pFastBac HTB载体。设计融合PCR引物，将mGoIL-17与GPV-VP3基因融合，并引入一段柔性Linker(G4S)3，将所获得的融合基因克隆至pFastBac HTB载体。将获得的重组转移载体质粒pFastBac HTB-mGoIL-17和pFastBacHTB-mGoIL17-VP3分别转化大肠杆菌DH10Bac感受态细胞，通过蓝白斑和三种抗生素（卡那霉素、四环素、庆大霉素）筛选，最终获得重组杆粒DNA。
参照FuGENE HD产品说明书将纯化的重组杆粒转染处于对数生长期的Sf9细胞，继续培养，直至出现明显细胞病变。收集培养上清，即为原代种毒（P1）。将P1代种毒以1:10的比例接种于对数生长期的单层Sf9细胞，27°C培养至90%的细胞出现病变时，收集培养上清，提取重组杆状病毒DNA进行PCR鉴定。将经过鉴定正确的重组杆状病毒P1代进行四次连续传代扩增，以提高重组杆状病毒的效价。应用SDS-PAGE及Western blot检测重组蛋白的表达。采用Ni-NTA His柱纯化的方法纯化mGoIL-17和mGoIL17-VP3重组蛋白。
制备鹅胚成纤维细胞，接种至6孔板中，分别加入1μg、5μg、10μg、20μg、50μg的mGoIL-17或者mGoIL17-VP3，以PBS作为对照。RT-PCR检测结果显示，以PBS为对照的中没有检测到鹅IL-6和IL-8的mRNA，而依次加入1-50μg的mGoIL-17/mGoIL17-VP3的试验组则可以检测到鹅IL-6和IL-8的mRNA。
4mGoIL-17黏膜佐剂效应的评估
从黑龙江省平山某养鹅场购进140只1月龄东北白鹅。共分7组，每组20只，分别点眼和滴鼻免疫VP3、IFN-α+VP3、LTB+VP3、mGoIL17+VP3、mGoIL17-VP3和PBS，肌肉注射商品化疫苗作为对照。每周每组采血（10份）分离血清以检测血清中免疫球蛋白（Ig）含量的变化和血清中和抗体效价。隔周每组杀东北白鹅（2只）分别取呼吸道和消化道灌洗液用以检测灌洗液中Ig的含量，分离外周血淋巴细胞用以检测CD4和CD8细胞的变化。结果显示，(1) mGoIL-17+VP3组和mGoIL17-VP3组与疫苗组有相似的抗体消长规律，在抗体的整体水平上疫苗组>mGoIL17-VP3组>mGoIL-17组>其它组，其中疫苗组、mGoIL17-VP3组和mGoIL-17组之间的差异不显著，均显著高于其他实验组；（2）各免疫组血清的中和抗体效价的变化趋势大体相同，在第7周抗体效价最高。其中mGoIL17-VP3组、mGoIL-17+VP3组、LTB组和疫苗组之间的差异不显著，均显著高于其他实验组；（3）间接ELISA检测呼吸道灌洗液中总Ig抗体，mGoIL-17+VP3组和mGoIL17-VP3组与其他组别有显著的差异性，说明mGoIL-17+VP3和mGoIL17-VP3在刺激呼吸道产生抗体的效果较明显；（4）间接ELISA检测消化道灌洗液中总Ig抗体，mGoIL-17+VP3组和mGoIL17-VP3组显著高于其他组别。说明mGoIL-17+VP3和mGoIL17-VP3在免疫远端的消化道也能起到刺激产生特异性抗体的作用；（5）分离的外周血淋巴细胞用细胞ELISA的方法检测各试验组间CD4+细胞含量的差异，IFN-α组、mGoIL-17+VP3组、mGoIL17-VP3组和疫苗组与对照组相比均能引起外周血淋巴细胞含量的增加，而LTB组不能引起显著的CD4+细胞含量的改变；（6）分离的外周血淋巴细胞用细胞ELISA的方法检测各试验组间CD8+细胞含量的差异，只有mGoIL-17+VP3组、mGoIL17-VP3组和疫苗组能引起外周血淋巴细胞含量的增加，而IFN-α组和LTB组不能引起显著的CD8+细胞含量的改变。
The mucosal surfaces of the gastrointestinal and respiratory tracts represent the principalportals of entry for most pathogens. Therefore the animal mucosal immune system plays a veryimportant role in the resistance to the invasion of pathogenic microorganisms. Effectivestimulation of the mucosal immune system needs to two basic conditions. First, the antigen in thevaccine can be effectively delivered to the mucosal lymphoid tissue; the second is a safe andeffective mucosal adjuvant to enhance the immune response. Immune adjuvant does not have theantigenicity, but it can promote vaccine antigen-specific immune response when incorporated intovaccine formulations. Until recently, IL-17-producing effector T helper cells, called Th17cells,have been discovered and characterized. IL17is an important and unique cytokine resistance tospecific pathogens. Because IL-17induces neutrophil trafficking to the site of inflammation, aswell as the promotion of the T cell response.
Relative to the study of mammalian mucosal immune, poultry mucosal immunity is relativelyslow, and most studies have focused on the study of the chicken. In this study, IL-17, CD4andCD8α gene of goose were cloned from thymocytes. The recombinant GoIL-17(rGoIL-17)expressed in baculovirus expression system was used as a mucosal immune adjuvant combinedwith goose parvovirus (GPV) VP3virus-like particles, or goose IL-17and VP3fusion expressionto immunize geese. The results showed that the goose IL-17as mucosal immune adjuvants caneffectively induce the body's mucosal immune and system immune. It has a very positive sense ofgosling plague prevention and the development of effective mucosal vaccine.
1cloning and sequence analysis of goose IL-17, CD4and CD8α gene
cDNA was synthesized with total RNA from goose thymus by using SMART (SwitchingMechanism at5’ End of RNA Transcript) Reverse Transcriptase. IL-17, CD4and CD8α specificprimers were designed according to the sequence of chicken IL-17(Genbank ID: NM_204460.1),duck CD4(Genbank ID: AF378701), and duck CD8α (Genbank ID: AF378373), which were usedto amplify the whole real sequence of goose IL-17with touchdown PCR. Amplified fragmentswere inserted into the pEASY-Blunt vector, and sent to the Genomics Institute for sequencing.
Sequences were initially analyzed using Blast search to confirm the correct gene was cloned.The sequences were submitted to NCBI and get a registration number, respectively. The CLUSTALW program was used to align the sequences and a phylogenetic tree was also created using the Neighbour-Joining method within the software MEGA5. The online software SignalP4.1Serverand TMHMM Server v.2.0were used to analyze signal peptide, transmembrane region predictionof IL-17, CD4and CD8α protein, in order to express mature extracellular region of goose IL-17(mGoIL-17) and geese CD4, CD8α.
2Prokaryotic expression of mGoIL-17and goose CD4, CD8α extracellular domain andpreparation of polyclonal antibody
According to signal peptide analysis of goose IL-17, and extracellular area analysis of gooseCD4and CD8α, the expression primers were designed. The PCR fragment was digested andligated into the pET32a expression vector. The vector was transformed into an E. coli Rosetta(DE3) pLysS strain, and the expression of recombinant protein was induced by IPTG. Therecombinant protein was purified with Ni-NTA His column. After purification, the recombinantprotein was immunized into rabbits to generate antiserum. The immunoreactivity of antiserum wasdetermined by Western blot. The antiserum was the foundation of the follow-up test.
3Eukaryotic expression and activity determination of the mGoIL-17and mGoIL-17fusionGPV-VP3protein
After digestion of pEASY-Blunt-mGoIL-17, the mGoIL-17fragment was ligated into thepFastBac HTB donor vector of the baculovirus expression system. The fusion fragment ofGPV-VP3, mGoIL-17, and the insertion of a flexible Linker (G4S)3was obtained by fusion PCRmethod. The fusion gene was sub-cloned into pFastBac HTB donor vector. The recombinantvectors were then transformed into DH10BAC bacterial cells. The final recombinant bacmidDNAs were obtained by blue and white, and three antibiotics (kanamycin, tetracycline, gentamicin)screening. Positive recombinant bacmid were used to transfect Sf9insect cells of logarithmicphase. All procedures were performed according to the manufacturer’s manual. The transfectedcells were continued to train until significant cytopathic. The culture supernatants were collected,namely, the primary generation virus (P1). P1virus was inoculated monolayer Sf9cells with1:10dilution, and cultured at27°C until90%CPE of the cells. The culture supernatants were collectedand the recombinant baculovirus DNA were extracted for identification by PCR. Recombinantbaculovirus underwent four rounds of amplification (72h each) by infecting Sf9monolayers togenerate of high titer recombinant virus. Proteins expression was analyzed on SDS-PAGE andwestern blot. The recombinant protein was purified with Ni-NTA His column.
The goose embryo fibroblasts were prepared from10-day-old goose embryo. Cells (1×107)were stimulated with1μg,5μg,10μg,20μg and50μg of rGoIL-17for12h in a DMEM medium.The PBS was used as control. The biological activities of rGoIL-17were assessed by RT-PCR. Theresults showed that, refold mGoIL-17/mGoIL17-VP3induced synthesis of IL-6and IL-8mRNArather than the control, and the expression of geese IL-6and IL-8was positively correlated withmGoIL-17/mGoIL17-VP3dose.
4Assessment of the immunity effect of mGoIL-17mucosal adjuvant
1401-month-old Northeast White Geese were purchased from a goose farm in pingshan ofHeilongjiang Province. The geese were divided into seven groups, n=20, respectively. The geesewere immunized with VP3, IFN-α+VP3, LTB+VP3, mGoIL17+VP3, fusion mGoIL17-VP3andPBS through eye and intranasal immunization, and immunized with the commercialization vaccinethrough intramuscular immunization as a positive control. Each set of blood (10parts) werecollected weekly to detect the titer changes of serum immunoglobulin (Ig) and neutralizingantibody. Two geese each group were killed every other week, and the respiratory andgastrointestinal lavage fluid were collected for detection of lavage Ig, and peripheral bloodlymphocytes were separated to detect the change of CD4and CD8cells. The results showed that,(1), mGoIL-17+VP3group mGoIL17-VP3group and the vaccine group had the similar antibodydynamic, and the overall level of the antibodies was vaccine group> mGoIL17-VP3group>mGoIL-17group> other groups;(2), The trend of neutralizing antibody titer of each group wassubstantially the same, as the highest antibody titer was in the7th week. mGoIL17-VP3andmGoIL-17+VP3group were much the same, and both higher than the vaccine group;(3), Total Igantibodies titer of respiratory lavage detected with indirect ELISA had significant differences withother groups, indicating that mGoIL-17+VP3and mGoIL17-VP3could obviously irritate theairways to produce antibodies;(4), Total Ig antibodies titer of gastrointestinal lavage detected withindirect ELISA had significant differences with other groups, indicating that mGoIL-17+VP3andmGoIL17-VP3could also play a role of stimuli of specific antibodies in immune distal digestivetract;(5), The differences of CD4+cell content among each group were tested by cell ELISA fromisolated peripheral blood lymphocyte cell. Compared with control group, IFN-α, mGoIL-17+VP3,mGoIL17-VP3, and the vaccine group could cause the increase of the content of peripheral bloodlymphocytes, but LTB group could not;(6), The differences of CD8+cell content among eachgroup were tested by cell ELISA from isolated peripheral blood lymphocyte cell. Compared withcontrol group, only mGoIL-17+VP3, mGoIL17-VP3, and the vaccine group could cause theincrease of the content of peripheral blood lymphocytes, but IFN-α and LTB group could not.
目前相对于哺乳动物黏膜免疫的研究，禽类黏膜免疫的研究相对缓慢，而且大部分研究都集中在鸡的相关研究。本研究首次克隆东北白鹅IL-17、CD4和CD8α基因，以真核表达的东北白鹅IL-17作为黏膜免疫佐剂与鹅细小病毒（GPV）的衣壳蛋白VP3病毒样粒子联合免疫，或东北白鹅IL-17与VP3融合表达后免疫东北白鹅，通过检测体液免疫和细胞免疫的多项指标对免疫效果进行评价，证实东北白鹅IL-17作为黏膜免疫佐剂可以有效诱导机体黏膜免疫和系统免疫，对小鹅瘟的防治及开发有效的黏膜疫苗具有十分积极的意义。
1东北白鹅IL-17、CD4和CD8α基因的克隆及序列分析
首先，从东北白鹅的胸腺组织中提取总RNA，经过SMART反转录酶合成双链cDNA，经PCR分别获得东北白鹅IL-17、CD4和CD8α基因，将扩增基因克隆至克隆载体并测序。
用DNAMAN6.03软件将测序的结果分别在美国国立生物技术信息中心（NCBI）的上进行Blast分析，与数据库中的序列进行相似性的比对。可以确定所克隆的基因为东北白鹅IL-17、CD4、CD8α基因，将序列提交至NCBI并获得登录号。确认克隆正确的东北白鹅IL-17、CD4和CD8α基因翻译成蛋白后用ClustalX2.1分别和NCBI中已经发表的各个物种的蛋白质序列进行蛋白序列同源性分析，然后再应用MEGA5.0生物软件程序以Neighbor-joining算法构建生物进化树。分别应用在线分析软件SignalP4.1Server和TMHMM Server v.2.0对东北白鹅IL-17、CD4和CD8α蛋白进行信号肽、跨膜区的预测分析，以便于进行下一步对成熟的东北白鹅IL-17（mGoIL-17）和东北白鹅CD4、CD8α胞外区的表达。
2mGoIL-17、东北白鹅CD4和CD8α胞外区原核表达及多克隆抗体的制备
根据东北白鹅IL-17的信号肽分析结果以及东北白鹅CD4和CD8α的胞外区的分析结果，设计表达引物扩增得到mGoIL-17、东北白鹅CD4和CD8α的胞外区，连接至pET-32a或pET-30a载体，然后转化至大肠杆菌Rosetta感受态细胞，IPTG诱导表达蛋白。采用Ni-NTAHis柱纯化方法纯化蛋白，免疫新西兰白兔制备多抗血清，通过western blot确定血清的反应性，为后续试验奠定基础。
3mGoIL-17和mGoIL-17融合GPV-VP3的真核表达及活性的测定
将pEASY-Blunt-mGoIL-17质粒通过双酶切得到IL17基因，克隆至pFastBac HTB载体。设计融合PCR引物，将mGoIL-17与GPV-VP3基因融合，并引入一段柔性Linker(G4S)3，将所获得的融合基因克隆至pFastBac HTB载体。将获得的重组转移载体质粒pFastBac HTB-mGoIL-17和pFastBacHTB-mGoIL17-VP3分别转化大肠杆菌DH10Bac感受态细胞，通过蓝白斑和三种抗生素（卡那霉素、四环素、庆大霉素）筛选，最终获得重组杆粒DNA。
参照FuGENE HD产品说明书将纯化的重组杆粒转染处于对数生长期的Sf9细胞，继续培养，直至出现明显细胞病变。收集培养上清，即为原代种毒（P1）。将P1代种毒以1:10的比例接种于对数生长期的单层Sf9细胞，27°C培养至90%的细胞出现病变时，收集培养上清，提取重组杆状病毒DNA进行PCR鉴定。将经过鉴定正确的重组杆状病毒P1代进行四次连续传代扩增，以提高重组杆状病毒的效价。应用SDS-PAGE及Western blot检测重组蛋白的表达。采用Ni-NTA His柱纯化的方法纯化mGoIL-17和mGoIL17-VP3重组蛋白。
制备鹅胚成纤维细胞，接种至6孔板中，分别加入1μg、5μg、10μg、20μg、50μg的mGoIL-17或者mGoIL17-VP3，以PBS作为对照。RT-PCR检测结果显示，以PBS为对照的中没有检测到鹅IL-6和IL-8的mRNA，而依次加入1-50μg的mGoIL-17/mGoIL17-VP3的试验组则可以检测到鹅IL-6和IL-8的mRNA。
4mGoIL-17黏膜佐剂效应的评估
从黑龙江省平山某养鹅场购进140只1月龄东北白鹅。共分7组，每组20只，分别点眼和滴鼻免疫VP3、IFN-α+VP3、LTB+VP3、mGoIL17+VP3、mGoIL17-VP3和PBS，肌肉注射商品化疫苗作为对照。每周每组采血（10份）分离血清以检测血清中免疫球蛋白（Ig）含量的变化和血清中和抗体效价。隔周每组杀东北白鹅（2只）分别取呼吸道和消化道灌洗液用以检测灌洗液中Ig的含量，分离外周血淋巴细胞用以检测CD4和CD8细胞的变化。结果显示，(1) mGoIL-17+VP3组和mGoIL17-VP3组与疫苗组有相似的抗体消长规律，在抗体的整体水平上疫苗组>mGoIL17-VP3组>mGoIL-17组>其它组，其中疫苗组、mGoIL17-VP3组和mGoIL-17组之间的差异不显著，均显著高于其他实验组；（2）各免疫组血清的中和抗体效价的变化趋势大体相同，在第7周抗体效价最高。其中mGoIL17-VP3组、mGoIL-17+VP3组、LTB组和疫苗组之间的差异不显著，均显著高于其他实验组；（3）间接ELISA检测呼吸道灌洗液中总Ig抗体，mGoIL-17+VP3组和mGoIL17-VP3组与其他组别有显著的差异性，说明mGoIL-17+VP3和mGoIL17-VP3在刺激呼吸道产生抗体的效果较明显；（4）间接ELISA检测消化道灌洗液中总Ig抗体，mGoIL-17+VP3组和mGoIL17-VP3组显著高于其他组别。说明mGoIL-17+VP3和mGoIL17-VP3在免疫远端的消化道也能起到刺激产生特异性抗体的作用；（5）分离的外周血淋巴细胞用细胞ELISA的方法检测各试验组间CD4+细胞含量的差异，IFN-α组、mGoIL-17+VP3组、mGoIL17-VP3组和疫苗组与对照组相比均能引起外周血淋巴细胞含量的增加，而LTB组不能引起显著的CD4+细胞含量的改变；（6）分离的外周血淋巴细胞用细胞ELISA的方法检测各试验组间CD8+细胞含量的差异，只有mGoIL-17+VP3组、mGoIL17-VP3组和疫苗组能引起外周血淋巴细胞含量的增加，而IFN-α组和LTB组不能引起显著的CD8+细胞含量的改变。
The mucosal surfaces of the gastrointestinal and respiratory tracts represent the principalportals of entry for most pathogens. Therefore the animal mucosal immune system plays a veryimportant role in the resistance to the invasion of pathogenic microorganisms. Effectivestimulation of the mucosal immune system needs to two basic conditions. First, the antigen in thevaccine can be effectively delivered to the mucosal lymphoid tissue; the second is a safe andeffective mucosal adjuvant to enhance the immune response. Immune adjuvant does not have theantigenicity, but it can promote vaccine antigen-specific immune response when incorporated intovaccine formulations. Until recently, IL-17-producing effector T helper cells, called Th17cells,have been discovered and characterized. IL17is an important and unique cytokine resistance tospecific pathogens. Because IL-17induces neutrophil trafficking to the site of inflammation, aswell as the promotion of the T cell response.
Relative to the study of mammalian mucosal immune, poultry mucosal immunity is relativelyslow, and most studies have focused on the study of the chicken. In this study, IL-17, CD4andCD8α gene of goose were cloned from thymocytes. The recombinant GoIL-17(rGoIL-17)expressed in baculovirus expression system was used as a mucosal immune adjuvant combinedwith goose parvovirus (GPV) VP3virus-like particles, or goose IL-17and VP3fusion expressionto immunize geese. The results showed that the goose IL-17as mucosal immune adjuvants caneffectively induce the body's mucosal immune and system immune. It has a very positive sense ofgosling plague prevention and the development of effective mucosal vaccine.
1cloning and sequence analysis of goose IL-17, CD4and CD8α gene
cDNA was synthesized with total RNA from goose thymus by using SMART (SwitchingMechanism at5’ End of RNA Transcript) Reverse Transcriptase. IL-17, CD4and CD8α specificprimers were designed according to the sequence of chicken IL-17(Genbank ID: NM_204460.1),duck CD4(Genbank ID: AF378701), and duck CD8α (Genbank ID: AF378373), which were usedto amplify the whole real sequence of goose IL-17with touchdown PCR. Amplified fragmentswere inserted into the pEASY-Blunt vector, and sent to the Genomics Institute for sequencing.
Sequences were initially analyzed using Blast search to confirm the correct gene was cloned.The sequences were submitted to NCBI and get a registration number, respectively. The CLUSTALW program was used to align the sequences and a phylogenetic tree was also created using the Neighbour-Joining method within the software MEGA5. The online software SignalP4.1Serverand TMHMM Server v.2.0were used to analyze signal peptide, transmembrane region predictionof IL-17, CD4and CD8α protein, in order to express mature extracellular region of goose IL-17(mGoIL-17) and geese CD4, CD8α.
2Prokaryotic expression of mGoIL-17and goose CD4, CD8α extracellular domain andpreparation of polyclonal antibody
According to signal peptide analysis of goose IL-17, and extracellular area analysis of gooseCD4and CD8α, the expression primers were designed. The PCR fragment was digested andligated into the pET32a expression vector. The vector was transformed into an E. coli Rosetta(DE3) pLysS strain, and the expression of recombinant protein was induced by IPTG. Therecombinant protein was purified with Ni-NTA His column. After purification, the recombinantprotein was immunized into rabbits to generate antiserum. The immunoreactivity of antiserum wasdetermined by Western blot. The antiserum was the foundation of the follow-up test.
3Eukaryotic expression and activity determination of the mGoIL-17and mGoIL-17fusionGPV-VP3protein
After digestion of pEASY-Blunt-mGoIL-17, the mGoIL-17fragment was ligated into thepFastBac HTB donor vector of the baculovirus expression system. The fusion fragment ofGPV-VP3, mGoIL-17, and the insertion of a flexible Linker (G4S)3was obtained by fusion PCRmethod. The fusion gene was sub-cloned into pFastBac HTB donor vector. The recombinantvectors were then transformed into DH10BAC bacterial cells. The final recombinant bacmidDNAs were obtained by blue and white, and three antibiotics (kanamycin, tetracycline, gentamicin)screening. Positive recombinant bacmid were used to transfect Sf9insect cells of logarithmicphase. All procedures were performed according to the manufacturer’s manual. The transfectedcells were continued to train until significant cytopathic. The culture supernatants were collected,namely, the primary generation virus (P1). P1virus was inoculated monolayer Sf9cells with1:10dilution, and cultured at27°C until90%CPE of the cells. The culture supernatants were collectedand the recombinant baculovirus DNA were extracted for identification by PCR. Recombinantbaculovirus underwent four rounds of amplification (72h each) by infecting Sf9monolayers togenerate of high titer recombinant virus. Proteins expression was analyzed on SDS-PAGE andwestern blot. The recombinant protein was purified with Ni-NTA His column.
The goose embryo fibroblasts were prepared from10-day-old goose embryo. Cells (1×107)were stimulated with1μg,5μg,10μg,20μg and50μg of rGoIL-17for12h in a DMEM medium.The PBS was used as control. The biological activities of rGoIL-17were assessed by RT-PCR. Theresults showed that, refold mGoIL-17/mGoIL17-VP3induced synthesis of IL-6and IL-8mRNArather than the control, and the expression of geese IL-6and IL-8was positively correlated withmGoIL-17/mGoIL17-VP3dose.
4Assessment of the immunity effect of mGoIL-17mucosal adjuvant
1401-month-old Northeast White Geese were purchased from a goose farm in pingshan ofHeilongjiang Province. The geese were divided into seven groups, n=20, respectively. The geesewere immunized with VP3, IFN-α+VP3, LTB+VP3, mGoIL17+VP3, fusion mGoIL17-VP3andPBS through eye and intranasal immunization, and immunized with the commercialization vaccinethrough intramuscular immunization as a positive control. Each set of blood (10parts) werecollected weekly to detect the titer changes of serum immunoglobulin (Ig) and neutralizingantibody. Two geese each group were killed every other week, and the respiratory andgastrointestinal lavage fluid were collected for detection of lavage Ig, and peripheral bloodlymphocytes were separated to detect the change of CD4and CD8cells. The results showed that,(1), mGoIL-17+VP3group mGoIL17-VP3group and the vaccine group had the similar antibodydynamic, and the overall level of the antibodies was vaccine group> mGoIL17-VP3group>mGoIL-17group> other groups;(2), The trend of neutralizing antibody titer of each group wassubstantially the same, as the highest antibody titer was in the7th week. mGoIL17-VP3andmGoIL-17+VP3group were much the same, and both higher than the vaccine group;(3), Total Igantibodies titer of respiratory lavage detected with indirect ELISA had significant differences withother groups, indicating that mGoIL-17+VP3and mGoIL17-VP3could obviously irritate theairways to produce antibodies;(4), Total Ig antibodies titer of gastrointestinal lavage detected withindirect ELISA had significant differences with other groups, indicating that mGoIL-17+VP3andmGoIL17-VP3could also play a role of stimuli of specific antibodies in immune distal digestivetract;(5), The differences of CD4+cell content among each group were tested by cell ELISA fromisolated peripheral blood lymphocyte cell. Compared with control group, IFN-α, mGoIL-17+VP3,mGoIL17-VP3, and the vaccine group could cause the increase of the content of peripheral bloodlymphocytes, but LTB group could not;(6), The differences of CD8+cell content among eachgroup were tested by cell ELISA from isolated peripheral blood lymphocyte cell. Compared withcontrol group, only mGoIL-17+VP3, mGoIL17-VP3, and the vaccine group could cause theincrease of the content of peripheral blood lymphocytes, but IFN-α and LTB group could not.
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