表达猪附红细胞体ENO蛋白的重组腺病毒对仔猪的免疫保护效力研究  

Studies on the immunoprotective efficacy of recombinant adenovirus expressing Mycoplasma suis ENO protein in piglets

作　　者：刘喆[1] 韩金成 王金琦 殷国璐 徐颖[1] 薛书江[1] 韩贞珍 LIU Zhe;HAN Jin-cheng;WANG Jin-qi;YIN Guo-lu;XU Ying;XUE Shu-jiang;HAN Zhen-zhen(Agricultural College of Yanbian University,Yanji 133002,China;Huludao Animal Epidemic Disease Control and Prevention Centre,Huludao 125000,China)

机构地区：[1]延边大学农学院,吉林延吉133002 [2]辽宁省葫芦岛市动物卫生防疫中心,辽宁葫芦岛125000

出　　处：《中国预防兽医学报》2024年第12期1270-1276,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：吉林省自然科学基金项目(YDZJ202201ZYTS616);国家自然科学基金项目(32360886)。

摘　　要：为评估表达猪附红细胞体α-烯醇化酶(ENO)蛋白的重组腺病毒对猪的免疫保护作用,本研究选取12头断奶仔猪均分为3组,A组经颈部肌肉注射1×1010pfu/mL的重组腺病毒2.5 mL,B组经颈部肌肉注射1×1010pfu/mL的重组空载体腺病毒2.5 mL,C组经颈部肌肉注射等量的PBS作为阴性对照组。共免疫两次,间隔21 d。在一免后每7 d收集仔猪血清,利用间接ELISA方法检测每组仔猪的抗体水平,为期49 d。二免后14 d,无菌摘取仔猪脾脏,制备脾淋巴细胞悬液,利用流式细胞术检测每组仔猪脾脏中的CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞的含量及CD4^(+)/CD8^(+)、CD3^(+)/CD4^(+)T淋巴细胞的比值。在摘脾7 d后利用纯化后的猪附红细胞体对仔猪攻毒并记录各组仔猪的临床症状。攻毒后14 d对所有仔猪经颈静脉采血,检测各组仔猪的血常规及血液生化指标。于攻毒后不同时间经颈静脉采血,采用PCR检测猪附红细胞体,确定感染时间及携带病原的持续时间,共观察49 d。结果显示,与B、C组相比,自免疫后28 d起,A组猪附红细胞体特异性抗体水平极显著升高(P<0.01)。流式细胞术检测结果显示,A组仔猪脾脏的CD3^(+)、CD4^(+)及CD8^(+)T淋巴细胞含量均极显著高于B、C组(P<0.01);A组仔猪CD4^(+)/CD8^(+)、CD3^(+)/CD4^(+)T淋巴细胞的比值均显著高于B、C组(P<0.05)。攻毒结果显示,B、C组仔猪出现相应发病症状且体温、体重、血液指标变化异常,A组仔猪未出现临床症状且各血液指标均在正常范围内。PCR结果显示,A组仔猪攻毒后携带病原持续时间为2d,B、C组仔猪攻毒后在整个实验周期49 d持续携带病原。综上所述,表达猪附红细胞体ENO蛋白的重组腺病毒能够对仔猪提供较强的抵抗强毒攻击的作用,对当前流行的猪附红细胞体病有良好的免疫保护效力。本研究为开发安全、高效的猪附红细胞体病疫苗提供了科学依据,并为深入了解ENO蛋白的免疫保护机�This study aimed to evaluate the immunoprotective effect of recombinant adenovirus expressing Mycoplasma suis ENO protein in pigs.Twelve weaned piglets were divided into three groups namely;group A was injected with 2.5mL of recombinant adenovirus at 1×1010pfu/mL via intramuscular injection in the neck;group B was injected with 2.5mL of recombinant empty vector adenovirus of 1×1010pfu/mL via neck muscle;and group C was injected with an equal amount of PBS via neck muscle as a negative control group.The piglets were immunized twice at an interval of 21 days.Serum samples were collected every 7 days after the first immunization,and the antibody level of each group was measured by indirect ELISA for 49 days.The antibody level of each group was measured by indirect ELISA at the end of the first immunization.14 days after the second immunization,spleens of piglets were harvested aseptically,and spleen lymphocyte suspensions were prepared,and then the contents of CD3^(+),CD4^(+)and CD8^(+)Tlymphocytes and the ratios of CD4^(+)/CD8^(+)and CD3^(+)/CD4^(+)T-lymphocytes were detected in the spleens of each group of piglets by flow cytometry.The purified Mycoplasma suis were used in a tetanus test on piglets 7 days after spleen removal,and the clinical symptoms of piglets in each group were recorded.Blood was collected from the jugular vein of all piglets 14 days after tetanus,and the blood routine and blood biochemical indexes of piglets in each group were examined.Blood was collected from the jugular vein at different times after the challenge,and PCR was used to detect Mycoplasma suis to determine the time of infection and the duration of carrying the pathogen.A total of 49 days were observed,and the results showed that the Mycoplasma suis-specific antibody levels in group A were significantly higher from the 28th days after immunization compared with those in groups B and C(P<0.01).Flow cytometry results showed that the contents of CD3^(+),CD4^(+)and CD8^(+)T-lymphocytes in the spleens of piglets in group A were extre

关 键 词：猪附红细胞体 重组腺病毒 仔猪 ENO 免疫保护 

分 类 号：S858.28[农业科学—临床兽医学]