马传染性贫血病毒囊膜蛋白GP90的表达及其免疫效果研究

Expression and immunogenicity of equine infectious anemia virus membrane protein GP90

目的 探索马传染性贫血病毒 (EIAV)野毒株 (强毒 ,LN)和疫苗毒株 (弱毒 ,DLV)的囊膜蛋白GP90作为鉴别诊断试剂的效果及基因工程疫苗的可能性。方法 将中国EIAV疫苗株 (DLV)及其亲本毒株辽毒株 (LN)接种于驴外周血白细胞培养物 ,提取前病毒DNA ,并以其为模板 ,经聚合酶链法 (PCR)扩增出LN和DLV的GP90片段 ,在Bac to Bac杆状病毒表达系统表达。表达的GP90蛋白经金属离子亲和层析 (IMAC)纯化后免疫小鼠 ,ELISA法检测抗EIAV抗体 ,中和试验检测中和抗体活性。同时对DLV和LNGP90的核苷酸与氨基酸进行比较。结果 DLV和LNGP90核苷酸序列的同源性为95 3 % ,氨基酸序列的同源性为 87.7%。未加佐剂组抗体滴度为 80 0 ,佐剂组抗体滴度达 160 0 ;不加佐剂组的中和抗体滴度在 40～ 80之间 ,加佐剂组中和抗体滴度在 80～ 160之间。结论 成功构建了分别表达EIAV野毒株LN和疫苗毒株DLV囊膜蛋白GP90的重组杆状病毒 ,大量表达的蛋白纯化后纯度较好 。

Objective Membrane protein GP90 of China equine infectious anemia virus (EIAV) vaccine strain (DLV) and its parental wild type LN strain were expressed with Bac to Bac baculovirus expression system and BALB/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine. Methods The authors infected donkey PBMC culture with China EIAV vaccine strain (DLV) and its parental wild type LN strain, extracted its proviral DNA as template, amplified the GP90 of DLV and LN, respectively, and expressed with Bac to Bac baculovirus expression system. The sf9 insect cells were infected with the recombinant baculovirus and the expressed proteins were purified by IMAC. BALB/c mice were inoculated with purified protein.The specific binding Abs generated in the immunized mice were determined by ELISA method. The neutralizing assay was set up to determine the neutralizing capability of the antigens generated in immunized animals. Results The recombinant virus expressing viral antigens was determined by Western blot. The expressed proteins were purified by IMAC resulting in the protein purity of 87%(DLV) and 82%(LN), respectively. The antibody titer of the groups with and without adjuvant was 1 600 and 800, respectively. Serial 2 fold dilutions of the immunized mice sera were reacted with 100 TCID50 of EIAV. The end point of immunization assay was to protect 50% cells form CPE caused by EIAV in donkey skin cells. The neutralizing antibody titer was in the range 40 to 80 from animal immunized with and without adjuvant. Conclusion Membrane proteins of EIAV vaccine strain and wild type strain were successfully expressed in eukaryotic cell expression system according to the scheduled plan. The proteins showed strong immunogenicity and could activate animals to produce anti EIAV specific antibody including neutralizing antibody to EIAV.