免疫刺激复合物在海水鱼的应用
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摘要
免疫刺激复合物（ISCOM）具有安全、高效、副作用小的优点，可作为免疫佐剂用于制备各类疫苗。目前，ISCOM主要在畜禽上应用，使用方式多为注射，由于ISCOM可经黏膜途径给药，因此很适于在鱼类口服和浸浴型疫苗上应用，目前还未见免疫刺激复合物在海水鱼上的应用报道。
本研究分别将BSA-ISCOM、创伤弧菌-ISCOM以及由创伤弧菌-ISCOM免疫蛋鸡而获得的卵黄粉应用于大黄鱼免疫，测定血清中超氧化物歧化酶（SOD）、补体C3（C3）、溶菌酶（LZM）、干扰素-γ（INF-γ）的含量或活力、血清抗体效价，分析由ISCOM技术制备的免疫制剂对大黄鱼免疫系统的影响。通过创伤弧菌攻毒，来评价ISCOM所介导的免疫保护率。
实验结果表明BSA-ISCOM免疫组第一次采集的血清中总蛋白浓度、SOD活力、溶菌酶、补体水平、干扰素γ水平与对照组相比差异显著（P<0.05），而第二次采集的血清中总蛋白浓度和溶菌酶与对照组差异显著（P<0.05）。免疫保护率为72.3%。
创伤弧菌-ISCOM免疫组第一次采集的血清中总蛋白浓度、血清抗体效价、SOD活力、干扰素γ与对照组相比差异显著（P<0.05），而第二次采集的血清中总蛋白浓度和抗体效价与对照组差异显著（P<0.05）。相对免疫保护率为88.4%。
卵黄抗体免疫组第一次采集的血清抗体效价、SOD活力、LZM浓度、干扰素γ水与对照组相比差异显著（P<0.05），而第二次采集的血清中抗体效价与对照组差异显著（P<0.05）。免疫保护率为46.7%。
同时，对含迟缓爱德华菌外膜蛋白OMPA，OMPS基因的重组菌进行了表达优化，提取纯化了OMPA，OMPS，制备了相应的免疫刺激复合物，为ISCOMs在海水鱼迟缓爱德华菌免疫防治中的应用打下了基础。
The immune stimulating complex(ISCOM),which can function as immuneadjuvant, is broadly used in producing vaccines for its safety, high-effectiveness andlow side-effect. Though the ISCOM is mainly applied by injection in immunizingpoultry,it can also be given via mucosal route, which makes it an ideal material tomanufacture vaccines for fish use given by feeding and steeping. Yet there's norelevant reports on the use of ISCOM in marine fish.
Based on technologies of producing ISCOM, three different immune stimulantswere specially made to immunize large yellow croaker(Pseudosciaena.crocea) in thisresearch:BSA-ISCOM,Vibrio.vulnificus-ISCOM and dried eggyolk produced byVibrio.vulnificus-ISCOM stimulating laying hens.And the impacts of these stimulantson the immune system of large yellow croaker were measured in terms of the activityof',superoxide,ismutase(SOD),complement,C3(C3),lysozyme(LZM),interferon-gamma(INF-γ) and antibody titer in the serum of test subjects.And,a pathogenic challengeposed by V.vulnificus was conducted on test subjects to evaluate the immuneprotection efficiency promoted by ISCOM.
Test results of blood samplings of test subjects immunized by BSA-ISCOMshowed that the activity of SOD,LZM,C3,INF-γ and total protein level in the serumwere all significantly higher than those of the control group during the firstsampling(P<0.05),while significant improvements were only observed in the activityof LZM and total protein level in the serum during the second sampling(P<0.05).Andthe immune protection rate of BSA-ISCOM on larger yellow croaker was72.3%.Besides, test results of blood samplings of test subjects immunized byVibrio.vulnificus-ISCOM showed that the activity of SOD,INF-γ and the level ofantibody titer and total protein in the serum were all significantly higher than those ofthe control group during the first sampling(P<0.05),and significant improvements inthe level of antibody titer and total protein in the serum were still observed during thesecond sampling(P<0.05).And immune protection rate of Vibrio.vulnificus-ISCOMon larger yellow croaker was88.4%.
And still, test results of blood samplings of test subjects immunized by driedeggyolk produced by Vibrio.vulnificus-ISCOM stimulating laying hens showed thatthe activity of SOD,LZM,INF-γ and the level of antibody titer in the serum were allsignificantly higher than those of the control group during the firstsampling(P<0.05),and significant improvement was only observed in the level ofantibody titer in the serum during the second sampling(P<0.05).And immuneprotection rate of dried eggyolk produced by Vibrio.vulnificus-ISCOM stimulatinglaying hens on larger yellow croaker was46.7%.
Meanwhile,the producing of outer membrane proteins OMPA and OMPS ofEdwardsiella.Tarda in Recombinant strains was facilitated.Furthermore,OMPA andOMPS were isolated,purified and made into related ISCOM,which laid thefoundation of the adoption of ISCOM in immunizing marine fish to E.tarda.
本研究分别将BSA-ISCOM、创伤弧菌-ISCOM以及由创伤弧菌-ISCOM免疫蛋鸡而获得的卵黄粉应用于大黄鱼免疫，测定血清中超氧化物歧化酶（SOD）、补体C3（C3）、溶菌酶（LZM）、干扰素-γ（INF-γ）的含量或活力、血清抗体效价，分析由ISCOM技术制备的免疫制剂对大黄鱼免疫系统的影响。通过创伤弧菌攻毒，来评价ISCOM所介导的免疫保护率。
实验结果表明BSA-ISCOM免疫组第一次采集的血清中总蛋白浓度、SOD活力、溶菌酶、补体水平、干扰素γ水平与对照组相比差异显著（P<0.05），而第二次采集的血清中总蛋白浓度和溶菌酶与对照组差异显著（P<0.05）。免疫保护率为72.3%。
创伤弧菌-ISCOM免疫组第一次采集的血清中总蛋白浓度、血清抗体效价、SOD活力、干扰素γ与对照组相比差异显著（P<0.05），而第二次采集的血清中总蛋白浓度和抗体效价与对照组差异显著（P<0.05）。相对免疫保护率为88.4%。
卵黄抗体免疫组第一次采集的血清抗体效价、SOD活力、LZM浓度、干扰素γ水与对照组相比差异显著（P<0.05），而第二次采集的血清中抗体效价与对照组差异显著（P<0.05）。免疫保护率为46.7%。
同时，对含迟缓爱德华菌外膜蛋白OMPA，OMPS基因的重组菌进行了表达优化，提取纯化了OMPA，OMPS，制备了相应的免疫刺激复合物，为ISCOMs在海水鱼迟缓爱德华菌免疫防治中的应用打下了基础。
The immune stimulating complex(ISCOM),which can function as immuneadjuvant, is broadly used in producing vaccines for its safety, high-effectiveness andlow side-effect. Though the ISCOM is mainly applied by injection in immunizingpoultry,it can also be given via mucosal route, which makes it an ideal material tomanufacture vaccines for fish use given by feeding and steeping. Yet there's norelevant reports on the use of ISCOM in marine fish.
Based on technologies of producing ISCOM, three different immune stimulantswere specially made to immunize large yellow croaker(Pseudosciaena.crocea) in thisresearch:BSA-ISCOM,Vibrio.vulnificus-ISCOM and dried eggyolk produced byVibrio.vulnificus-ISCOM stimulating laying hens.And the impacts of these stimulantson the immune system of large yellow croaker were measured in terms of the activityof',superoxide,ismutase(SOD),complement,C3(C3),lysozyme(LZM),interferon-gamma(INF-γ) and antibody titer in the serum of test subjects.And,a pathogenic challengeposed by V.vulnificus was conducted on test subjects to evaluate the immuneprotection efficiency promoted by ISCOM.
Test results of blood samplings of test subjects immunized by BSA-ISCOMshowed that the activity of SOD,LZM,C3,INF-γ and total protein level in the serumwere all significantly higher than those of the control group during the firstsampling(P<0.05),while significant improvements were only observed in the activityof LZM and total protein level in the serum during the second sampling(P<0.05).Andthe immune protection rate of BSA-ISCOM on larger yellow croaker was72.3%.Besides, test results of blood samplings of test subjects immunized byVibrio.vulnificus-ISCOM showed that the activity of SOD,INF-γ and the level ofantibody titer and total protein in the serum were all significantly higher than those ofthe control group during the first sampling(P<0.05),and significant improvements inthe level of antibody titer and total protein in the serum were still observed during thesecond sampling(P<0.05).And immune protection rate of Vibrio.vulnificus-ISCOMon larger yellow croaker was88.4%.
And still, test results of blood samplings of test subjects immunized by driedeggyolk produced by Vibrio.vulnificus-ISCOM stimulating laying hens showed thatthe activity of SOD,LZM,INF-γ and the level of antibody titer in the serum were allsignificantly higher than those of the control group during the firstsampling(P<0.05),and significant improvement was only observed in the level ofantibody titer in the serum during the second sampling(P<0.05).And immuneprotection rate of dried eggyolk produced by Vibrio.vulnificus-ISCOM stimulatinglaying hens on larger yellow croaker was46.7%.
Meanwhile,the producing of outer membrane proteins OMPA and OMPS ofEdwardsiella.Tarda in Recombinant strains was facilitated.Furthermore,OMPA andOMPS were isolated,purified and made into related ISCOM,which laid thefoundation of the adoption of ISCOM in immunizing marine fish to E.tarda.
引文
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