抗硬蜱亚单位疫苗用候选抗原蛋白的筛选及免疫原性分析  

Screening and immunogenicity analysis of candidate antigenic proteins for ixodes subunit vaccine

作　　者：刘燕 师光鑫 甘露 何文文 伍军 颜成旭 呼尔查[1] 温丽翠 李才善 郭庆勇[1] 巴音查汗·盖力克 LIU Yan;SHI Guangxin;GAN Lu;HE Wenwen;WU Jun;YAN Chengxu;HU Ercha;WEN Licui;LI Caishan;GUO Qingyong;BAYINCHAHAN Gailike(College of Veterinary Medicine,Xinjiang Agricultural University,Urumuqi 830052,China;Xinjiang Center of Animal Disease Control,Urumqi 830000,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]新疆动物卫生监督所,新疆乌鲁木齐830000

出　　处：《中国兽医学报》2024年第3期519-526,共8页Chinese Journal of Veterinary Science

基　　金：新疆农业大学研究生科研创新计划资助项目(XJAUGRI2023004);新疆维吾尔自治区科技厅-国际合作专项资助项目(2021E01001)。

摘　　要：对已经构建保存的PET-28a-GSTK1、PET-28a-GSTE3、PET-28a-Fe1、PET-28a-Fe2和PET-28a-CPL质粒,利用PCR技术检测目的基因,经IPTG诱导后通过SDS-PAGE检测5种重组蛋白(rGSTK1、rGSTE3、rFe1、rFe2和rCPL)的表达,利用His标签蛋白纯化试剂盒纯化4种重组蛋白(rGSTK1、rFe1、rFe2和rCPL),并使用BCA蛋白定量测定试剂盒测定蛋白浓度后,免疫BALB/c小鼠制备多克隆抗体,经Western blot测定4种重组蛋白的反应原性,采用间接Elisa测定多克隆抗体效价以及免疫后抗体消长规律。结果显示,5种目的基因条带正确,并未发生丢失;SDS-PAGE检测发现除rGSTE3外,其余4种蛋白(rGSTK1、rFe1、rFe2和rCPL)均能稳定表达且大小分别为28、25、26、27 kDa,以包涵体形式表达;利用His标签纯化蛋白后测定蛋白浓度,结果显示,以250 mmol/L咪唑进行洗脱时可获得高纯度的目的蛋白,蛋白质量浓度分别为2.633、2.532、2.582和2.506 g/L;将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体,间接ELISA结果显示,4种重组蛋白(rGSTK1、rFe1、rFe2和rCPL)制备的多克隆抗体效价分别高达1∶819200、1∶819200、1∶1638400和1∶409600,表明4种重组蛋白的免疫原性较好,其中rFe2的免疫原性最好;三免后的21 d 4种重组蛋产生的抗体均达到峰值后逐渐降低,但三免后的63 d抗体水平仍处于较高水平,其中rFe2的抗体水平最高,为1∶819200;rGSTK1次之,为819200。Western blot结果显示,4种重组蛋白均可与其多克隆抗体发生特异性反应;其中rFe2的反应原性最强,rGSTK1次之。本研究以重组蛋白的稳定性、高表达、免疫后抗体水平以及与多克隆抗体反应原性为指标,筛选出rFe2为最佳候选抗原蛋白,另外rGSTK1效果与其相当,也可以考虑2种蛋白联合使用,为后续抗硬蜱疫苗的研发提供试验基础。The aim of this study is to screen the best candidate antigenic proteins for ixodes subunit vaccine.In this study,the target genes in PET-28 a-GSTK1,PET-28a-GSTE3,PET-28a-Fe1,PET-28a-Fe2 and PET-28a-CPL plasmids which had been constructed previously were detected by PCR.The expression of five recombinant proteins(rGSTK1,rGSTE3,rFel,rFe2 and rCPL)was detected by SDS-PAGE after IPTG induction,and the four recombinant proteins(rGSTK1,rFe1,rFe2and rCPL)were purified by His-tagged protein purification kit,and the protein concentrations were determined by BCA protein quantification kit.The reactivity of the four recombinant proteins was determined by Western blot,and the potency of the polyclonal antibodies and the pattern of antibody elongation after immunization were determined by indirect Elisa.The results showed that the five target gene bands were correct and did not lose.SDS-PAGE showed that except for rGSTE3,the other four proteins(rGSTK1,rFe1,rFe2 and rCPL)were stably expressed in the form of inclusion bodies with the sizes of 28,25,26,27 kDa,respectively.His label was used to purify the protein and determine the protein concentration.The results showed that the target protein with high purity could be obtained by elution with 250 mmol/L imidazole,and the protein concentrations were 2.633,2.532,2.582 and 2.506 g/L,respectively.The purified recombinant protein was used to immunize BALB/c mice to prepare polyclonal antibodies.Indirect ELISA results showed that the titers of the four recombinant proteins(rGSTK 1,rFe1,rFe2 and rCPL)were as high as1:819200,1:819200,respectively 1:1638400 and 1:409600,indicating that the immunogenicity of the four recombinant proteins was good,rFe2 had the best immunogenicity.The antibody produced by the four recombinant eggs reached the peak at 21 days after triple immunization and gradually decreased,but the antibody level at 63 days after triple immunization was still at a high level,with rFe2 having the highest antibody level at 1:819200;RGSTK1 came second at 819200.Western blot

关 键 词：抗硬蜱亚单位疫苗 抗原蛋白 多克隆抗体 免疫原性 

分 类 号：S855.9[农业科学—临床兽医学]