本文主要研究内容
作者张海朝（2019）在《鸡柔嫩艾美耳球虫多聚体疫苗EtDLC8a-GST-MIC2的构建与免疫保护效果的评价》一文中研究指出：在生物体中,蛋白质的同源二聚结构是一种普遍存在的现象。将两种具有同源二聚结构的蛋白融合后可形成具有线性结构或网状结构的同源多聚体。该同源多聚体应用范围广泛,其中便可作为呈递抗原蛋白的疫苗平台,通过插入不同的抗原表位,提高抗原载量,刺激机体产生更好的免疫保护效果。本研究中以该同源多聚体疫苗平台为思路,应用基因工程技术将扩增的柔嫩艾美耳球虫的动力蛋白轻链8a(EtDLC8a)基因克隆到含有谷胱甘肽巯基转移酶(GST)基因的PGEX-6P-1载体上,构建了EtDLC8a-GST疫苗平台。经Native-PAGE和Western blot验证构建的EtDLC8a-GST可形成多聚体。选取EtDLC8a基因中非结构区域作为抗原片段插入位点,对EtDLC8a-GST疫苗平台进一步改造完善。同时,选取柔嫩艾美耳球虫抗原MIC2部分抗原片段,插入到疫苗平台中,构建了EtDLC8a-GST-MIC2多聚蛋白复合体疫苗,并对该多聚蛋白复合体疫苗进行了保护性评价。通过Native-PAGE和Western-Blot技术分析表明,选取构建的外源片段插入位点不会影响EtDLC8a-GST疫苗平台形成的多聚结构,同时将抗原片段EtMIC2插入疫苗平台后对多聚结构的形成不会造成影响。通过动物试验对重组疫苗进行了免疫保护效果的评价。将210只一日龄海兰褐公雏鸡随机分7组,即不免疫不感染组(I组)、不免疫感染组(II组)、EtMIC2抗原片段免疫组(III组)、TgDLC8a-GST免疫组(IV组)、EtDLC8a-GST免疫组(V组)、TgDLC8a-GST-MIC2免疫组(VI组)和EtDLC8a-GST-MIC2免疫组(VII组)。对雏鸡进行7日龄初免,14日龄二免,在21日龄时除不免疫不感染组(I组)外其余组均进行攻虫感染。通过体重增长、盲肠病变、卵囊排出量、淋巴因子水平、血清及粘膜抗体水平等对免疫保护效果进行评价。动物试验结果表明,EtMIC2抗原片段免疫组(III组)(ACI=92.70)与对照组比较,部分抗原片段所提供的免疫保护力相对较弱。TgDLC8a-GST-MIC2免疫组(VI组)(ACI=137.00)和EtDLC8a-GST-MIC2免疫组(VII组)(ACI=136.44)与EtMIC2抗原片段免疫组(III组)(ACI=92.70)比较说明,多聚复合物提供的免疫保护效果优于蛋白单体(ACI评价标准:ACI<120为无效,120<ACI<160为中等有效,160<ACI<180为良好,ACI>180为优秀)。血清抗体与粘膜抗体水平结果表明,EtMIC2抗原片段免疫组(III组)、TgDLC8a-GST-MIC2免疫组(VI组)和EtDLC8a-GST-MIC2免疫组(VII组)与对照组相比差异显著(P<0.05)。TgDLC8a-GST-MIC2免疫组(VI组)和EtDLC8a-GST-MIC2免疫组(VII组)与EtMIC2抗原片段免疫组(III组)相比差异显著(P<0.05)。在细胞因子水平方面,TgDLC8a-GST-MIC2免疫组(VI组)和EtDLC8a-GST-MIC2免疫组(VII组)与EtMIC2抗原片段免疫组(III组)相比,具有显著性差异(P<0.05)。另外,淋巴细胞亚群结果表明,在CD4~+与CD8~+T淋巴细胞亚群水平,TgDLC8a-GST-MIC2免疫组(VI组)和EtDLC8a-GST-MIC2免疫组(VII组)与EtMIC2抗原片段免疫组(III组)相比,具有显著性差异(P<0.05)。综上,应用多聚体疫苗平台可提升部分抗原的免疫保护作用。
Abstract
zai sheng wu ti zhong ,dan bai zhi de tong yuan er ju jie gou shi yi chong pu bian cun zai de xian xiang 。jiang liang chong ju you tong yuan er ju jie gou de dan bai rong ge hou ke xing cheng ju you xian xing jie gou huo wang zhuang jie gou de tong yuan duo ju ti 。gai tong yuan duo ju ti ying yong fan wei an fan ,ji zhong bian ke zuo wei cheng di kang yuan dan bai de yi miao ping tai ,tong guo cha ru bu tong de kang yuan biao wei ,di gao kang yuan zai liang ,ci ji ji ti chan sheng geng hao de mian yi bao hu xiao guo 。ben yan jiu zhong yi gai tong yuan duo ju ti yi miao ping tai wei sai lu ,ying yong ji yin gong cheng ji shu jiang kuo zeng de rou nen ai mei er qiu chong de dong li dan bai qing lian 8a(EtDLC8a)ji yin ke long dao han you gu guang gan tai qiu ji zhuai yi mei (GST)ji yin de PGEX-6P-1zai ti shang ,gou jian le EtDLC8a-GSTyi miao ping tai 。jing Native-PAGEhe Western blotyan zheng gou jian de EtDLC8a-GSTke xing cheng duo ju ti 。shua qu EtDLC8aji yin zhong fei jie gou ou yu zuo wei kang yuan pian duan cha ru wei dian ,dui EtDLC8a-GSTyi miao ping tai jin yi bu gai zao wan shan 。tong shi ,shua qu rou nen ai mei er qiu chong kang yuan MIC2bu fen kang yuan pian duan ,cha ru dao yi miao ping tai zhong ,gou jian le EtDLC8a-GST-MIC2duo ju dan bai fu ge ti yi miao ,bing dui gai duo ju dan bai fu ge ti yi miao jin hang le bao hu xing ping jia 。tong guo Native-PAGEhe Western-Blotji shu fen xi biao ming ,shua qu gou jian de wai yuan pian duan cha ru wei dian bu hui ying xiang EtDLC8a-GSTyi miao ping tai xing cheng de duo ju jie gou ,tong shi jiang kang yuan pian duan EtMIC2cha ru yi miao ping tai hou dui duo ju jie gou de xing cheng bu hui zao cheng ying xiang 。tong guo dong wu shi yan dui chong zu yi miao jin hang le mian yi bao hu xiao guo de ping jia 。jiang 210zhi yi ri ling hai lan he gong chu ji sui ji fen 7zu ,ji bu mian yi bu gan ran zu (Izu )、bu mian yi gan ran zu (IIzu )、EtMIC2kang yuan pian duan mian yi zu (IIIzu )、TgDLC8a-GSTmian yi zu (IVzu )、EtDLC8a-GSTmian yi zu (Vzu )、TgDLC8a-GST-MIC2mian yi zu (VIzu )he EtDLC8a-GST-MIC2mian yi zu (VIIzu )。dui chu ji jin hang 7ri ling chu mian ,14ri ling er mian ,zai 21ri ling shi chu bu mian yi bu gan ran zu (Izu )wai ji yu zu jun jin hang gong chong gan ran 。tong guo ti chong zeng chang 、mang chang bing bian 、luan nang pai chu liang 、lin ba yin zi shui ping 、xie qing ji nian mo kang ti shui ping deng dui mian yi bao hu xiao guo jin hang ping jia 。dong wu shi yan jie guo biao ming ,EtMIC2kang yuan pian duan mian yi zu (IIIzu )(ACI=92.70)yu dui zhao zu bi jiao ,bu fen kang yuan pian duan suo di gong de mian yi bao hu li xiang dui jiao ruo 。TgDLC8a-GST-MIC2mian yi zu (VIzu )(ACI=137.00)he EtDLC8a-GST-MIC2mian yi zu (VIIzu )(ACI=136.44)yu EtMIC2kang yuan pian duan mian yi zu (IIIzu )(ACI=92.70)bi jiao shui ming ,duo ju fu ge wu di gong de mian yi bao hu xiao guo you yu dan bai chan ti (ACIping jia biao zhun :ACI<120wei mo xiao ,120<ACI<160wei zhong deng you xiao ,160<ACI<180wei liang hao ,ACI>180wei you xiu )。xie qing kang ti yu nian mo kang ti shui ping jie guo biao ming ,EtMIC2kang yuan pian duan mian yi zu (IIIzu )、TgDLC8a-GST-MIC2mian yi zu (VIzu )he EtDLC8a-GST-MIC2mian yi zu (VIIzu )yu dui zhao zu xiang bi cha yi xian zhe (P<0.05)。TgDLC8a-GST-MIC2mian yi zu (VIzu )he EtDLC8a-GST-MIC2mian yi zu (VIIzu )yu EtMIC2kang yuan pian duan mian yi zu (IIIzu )xiang bi cha yi xian zhe (P<0.05)。zai xi bao yin zi shui ping fang mian ,TgDLC8a-GST-MIC2mian yi zu (VIzu )he EtDLC8a-GST-MIC2mian yi zu (VIIzu )yu EtMIC2kang yuan pian duan mian yi zu (IIIzu )xiang bi ,ju you xian zhe xing cha yi (P<0.05)。ling wai ,lin ba xi bao ya qun jie guo biao ming ,zai CD4~+yu CD8~+Tlin ba xi bao ya qun shui ping ,TgDLC8a-GST-MIC2mian yi zu (VIzu )he EtDLC8a-GST-MIC2mian yi zu (VIIzu )yu EtMIC2kang yuan pian duan mian yi zu (IIIzu )xiang bi ,ju you xian zhe xing cha yi (P<0.05)。zeng shang ,ying yong duo ju ti yi miao ping tai ke di sheng bu fen kang yuan de mian yi bao hu zuo yong 。
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