LMH细胞的悬浮驯化及其在研发禽腺病毒8型候选疫苗中的应用  

Suspension domestication of LMH cells and its application in the development of avian adenovirus type 8 vaccine candidates

作　　者：高艳[1,2] 都兴洋 郭茹 潘青 张峣 杨帆 张宇[1] 刘景利 秦运安[1] 祁小乐[1] 高玉龙 刘慧敏[1,2] 高宏雷[1] GAO Yan;DU Xing-yang;GUO Ru;PAN Qing;ZHANG Yao;YANG Fan;ZHANG Yu;LIU Jing-li;QIN Yun-an;QI Xiao-le;GAO Yu-long;LIU Hui-min;GAO Hong-lei(Avian Immunosuppressive Disease Innovation Team,National Key Laboratory for Animal Disease Prevention and Control,Heilongjiang Province Veterinary Biological Medicine Engineering Technology Research Center,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;Harbin Weike Biotechnology Co.,Ltd.,Harbin 150069,China;Huangpu Customs Technology Center,Guangzhou 510700,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室/禽免疫抑制病创新团队/黑龙江省兽用生物药物工程技术研究中心,黑龙江哈尔滨150069 [2]哈尔滨维科生物技术有限公司,黑龙江哈尔滨150069 [3]黄埔海关技术中心,广东广州510700

出　　处：《中国预防兽医学报》2024年第12期1277-1283,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家肉鸡产业技术体系(CARS-41)。

摘　　要：为实现禽腺病毒8b型(FAdV-8b)的无血清悬浮细胞的培养工艺,并评价利用该工艺培养FAdV-8b制备的灭活疫苗的免疫效力,本研究采用贴壁的鸡肝癌上皮细胞(LMH细胞)逐步降低培养基中血清含量的方法,筛选适应低血清生长的贴壁细胞株,将其驯化成无血清悬浮生长的LMH细胞,命名为LMH-S。结果显示,经无血清驯化后的悬浮LMH-S细胞增殖稳定,存活率在90%以上。优化摇瓶的接毒工艺,确定摇瓶悬浮培养FAdV-8b工艺为1‰体积比接种病毒,96 h收获病毒。将LMH-S细胞接入Eppendon DASGIP 4联平行生物反应器中培养72 h后,按照体积比1‰接入FAdV-8b,96 h收获病毒测定病毒含量。结果显示,生物反应器中制备的FAdV-8b效价可达10^(8.5)TCID_(50)/mL,利用其制备的灭活疫苗以1 mL/只剂量免疫2周龄SPF鸡评估该疫苗的安全性。以0.5 mL/只免疫2周龄SPF鸡,于免疫后7 d、14 d分别采血,采用ELISA方法检测各组鸡血清中的抗体水平,于免疫后14 d攻击FAdV-8b强毒株。观察攻毒后各组鸡的临床症状及各脏器的剖检变化,采集肝脏病料样品制备病理切片,采用荧光定量PCR方法检测鸡肝脏中的病毒载量。结果显示疫苗的安全性良好。抗体检测结果显示,免疫后7 d抗体无转阳,免疫后14 d抗体的阳转率为73.3%(11/15)。攻毒后4 d剖检免疫组鸡的肝脏均正常;对照组鸡肝脏均有肿大,有的肝脏呈土黄色,质脆,个别有点状出血;病理切片观察可见,免疫组鸡肝细胞仅有一定程度变性,对照组鸡肝细胞广泛变性、局部肝细胞坏死并伴有大量炎性细胞浸润。病毒载量检测结果显示,攻毒后4 d对照鸡肝脏病毒载量极显著高于免疫组鸡(P<0.0001),攻毒后14 d对照组鸡肝脏的病毒载量仍高于免疫组(P>0.05)。攻毒后14 d所有组鸡肝脏均无明显变化。上述结果表明通过悬浮培养LMH-S细胞制备的FAdV-8b灭活疫苗安全性良好,免疫后可以诱导鸡产生较强的体液免疫�In order to achieve serum-free suspension cell culture of avian adenovirus 8 type b(FAdV-8b),and to use this technology to culture FAdV-8b to prepare inactivated vaccine to immunize SPF chicks,and evaluate the immune efficacy of the vaccine,this study adopted the method of gradually reducing the serum content in the culture medium of adherent chicken hepatocellular carcinoma epithelial cells(LMH cells)to obtain an adherent cell line adapted to low serum growth,and then domesticated into serum-free suspension growth of LMH cells,named LMH-S.The results showed that the proliferation of suspended LMH-S cells after serum-free acclimation was stable and the survival rate was above 90%.The technology of FAdV-8b in flask culture was explored.The process of FAdV-8b suspension culture was determined to inoculated the virus at a volume ratio of 1‰dose,and the virus was harvested 96 hours after inoculation.LMH-S cells were added to Eppendon DASGIP 4-parallel bioreactor,and cultured for 72 hours.FAdV-8b was inoculated at a volume ratio of 1‰and harvested at 96 hours after inoculation,and the virus content was measured.The results showed that the titer of FAdV-8b prepared in the bioreactor could reach 10^(8.5)TCID_(50)/mL.The inactivated vaccine was prepared and the safety of the vaccine was evaluated by immunizing 2-week-old SPF chickens at a dose of 1mL per chicken.2-week-old SPF chickens were immunized with 0.5mL per chicken,and blood samples were collected at 7 days and 14 days after immunization.The antibody level of chickens in each group was detected by ELISA method,and the FAdV-8b strain was used to chanlleng 14 days after immunization.The clinical symptoms and necropsy changes of chickens in each group were observed,liver disease materials were collected to prepare pathological sections,and viral load in the liver was detected by fluorescence quantitative PCR.The results showed that the vaccine was safe.The antibody test results showed that the antibody did not turn positive on the 7th day after immunization,and

关 键 词：LMH细胞 悬浮驯化 禽腺病毒8型 

分 类 号：S852.65[农业科学—基础兽医学]