Development Of Tetravalent Recombinant Subunit Vaccine For Coccidia In Chicken

Chicken coccidiosis is an acute epidemic parasitic protozoan disease caused by one or more Eimeria species throughout the world.It is one of the major diseases with a highly negative impact on chicken production in China.Eimeria tenella(E.tenella),Eimeria necatrix(E.necatrix),Eimeria maxima(E.maxima)and Eimeria acervuline(E.acervulina)are mostly mixed infections.At present,the prevention and control measures for chicken coccidiosis mainly depend on drug and live oocyst vaccine.While the live oocyst vaccine has the risk of virulence reversion,detoxification,complicated production process and high cost.Therefore,it is urgent to develop a safe and stable vaccine.The genetic engineering vaccine constructed by modern biotechnology has been widely concerned because of good safety,no reversion and low cost.In recent years,although many scholars devote themselves to the development of genetic engineering vaccines against chicken coccidiosis,there is no commercialized genetic engineering vaccine in domestic.Moreover,chicken coccidiosis is mostly caused by several coccidia together,the single vaccine couldn’t meet the prevention and control for chicken coccidiosis.So the vaccine which can resist multiple coccidia infection is needed.Selecting the protective antigen genes is the key to study genetic engineering vaccine.The protective antigens of chicken coccidia in the sporozoite,merozoite and gametophyte stages have good immunogenicity and can induce a protective immune response.Moreover,the expression vector systems mainly include E.coli,bacillus subtilis,Bacillus Calmette-Guerin(BCG),lactic acid bacteria and yeast etc.Pichia pastoris expression system has been widely concerned because of the simple operation,fast growth,high protein expression and low cost.The single or multiple coccidia genetic engineering vaccines have been reported.Recombination of different coccidia genes can prevent multiple coccidia infections and reduce their production processes.Many studies have shown that the multivalent recombinant vaccine and nucleic acid vaccine expressed by E.coli system have certain immune protection effects,the research on the preparation of tetravalent recombinant subunit vaccine by tandem expression of different chicken coccidiosis genes using Pichia pastoris system has not been reported.Many studies showed that the immune protection effect could be achieved by immunizing chickens with different coccidia antigen genes induced by E.coli and Pichia pastoris system.However,the research on the development of tetravalent recombinant subunit vaccine using chicken coccidia antigen genes in series with Pichia pastoris system has not been reported.If the tetravalent recombinant protein expressed by Pichia pastoris system can play a better immune protection effect after immunizing animals,compared with the mixed immunization of four recombinant proteins,it not only optimizes its production process,but also greatly improves the production efficiency.Therefore,in this study,the four genes including E.tenella TA4(Et TA4),E.acervulina 3-1E(Ea3-1E),E.maxima IMP1(Em IMP1)and E.necatrix NA4(En NA4)with good immune protection in sporozoite and merozoite stages were amplified and connected by GS Linker(named TEIN).TEIN was inserted into the Pichia pastoris p PIC9K expression vector and induced by GS115strain.At the same time,E.coli expression system was used as control.After the14-day-old chickens were immunized with the purified recombinant protein,the anticoccidial index ACI,the optimal immunization dose,immune duration,storage period and safety were comprehensively evaluated.The concrete research contents are as follows:Expression of four recombinant genes of chicken coccidia by Pichia pastoris and E.coli systems:Et TA4,Ea3-1E,Em IMP1 and En NA4 genes were amplified from the c DNA of E.tenella,E.acervulina,E.maxima and E.necatrix respectively.The four genes were connected in series by GS Linker(named TEIN).The recombinant expression plasmids p PIC9K-TA4,p PIC9K-NA4,p PIC9K-3-1E,p PIC9K-IMP1 and p PIC9K-TEIN were constructed and then transformed into Pichia pastoris GS115,induced by BMGY and BMMY medium.The expression plasmids p ET32a-TA4,p ET32a-NA4,p ET32a-3-1E,p ET32a-IMP1 and p ET32a-TEIN were constructed and then transformed into E.coli BL21 induced by IPTG,which compared with the Pichia pastoris expression system.The results showed that Et TA4(540 bp),Ea3-1E(510bp),Em IMP1(1128bp),En NA4(543 bp)and TEIN(2900 bp)genes were successfully amplified.The target proteins were identified by SDS-PAGE(excluding vector tags)as 20 KD,19 KD,42 KD,20 KD and 101 KD.Western blot results showed that all recombinant proteins had good reactogenicity.Determination on the optimal immune dose of tetravalent recombinant subunit vaccine for coccidia in chicken:At 14 days of age,the recombinant subunit vaccine was intramuscularly immunized with the high(150μg/feather),medium(100μg/feather)and low(50μg/feather)dose respectively.The same dose was inoculated on 21 days old,then after 7 days(28 days of age)10,000 sporulated oocysts of different coccidia were administered.The anticoccidial index ACI was evaluated by survival rate,relative weight gain rate,intestinal lesion score and OPG.The levels of specific antibodies Ig G,IL-2,IL-10 and IFN-γin serum were detected by indirect ELISA.The number of T lymphocytes in spleen was measured by flow cytometry.All the results showed that the Ig G titer of the middle dose group(100μg/feather)was significantly different from the first immunization(p<0.05).The level of IL-2 in the high,medium and low dose groups increased significantly after booster immunization(p<0.05).the IL-2 titer reached 380 pg/ml after the second immunization in the medium dose group.While the level of IL-10 did not change significantly in the three groups(p>0.05);the titer of IFN-γwas up to 314pg/ml after immunization with 100μg/feather.The proportion of CD4~+and CD8~+T cells was significantly different from the control group(p<0.05),the proportion of CD4~+T cells in the middle dose group was significantly different between the two immunizations(p<0.05).In all experimental groups,the ACI of middle dose group(100μg/feather)was up to 180.61.Therefore,100μg/feather was used as the optimal immune dose for the tetravalent recombinant subunit vaccine,which laid a foundation for the immune protection effect.The immune protection of tetravalent recombinant subunit vaccine for coccidia in chicken:The recombinant proteins were mixed with aluminum hydroxide adjuvant(1:1)to prepare subunit vaccines and inoculated intramuscularly with100μg/feather on 14 days of age.The immunization groups were divided into Pichia pastoris and E.coli expression groups including mixed immunization and TEIN protein group respectively.The live oocyst vaccine used as control.The immunization times included single and booster.The single immunization group was intramuscularly injected with recombinant subunit vaccine at 100μg/feather on 14d.At 21 days of age,except for the PBS group,the other groups were administered with10,000 different sporulated oocysts.The booster immunization groups were intramuscularly inoculated with 100μg/feather at 14,21d and challenged at 28d.Five chickens were randomly selected at 35d from each group,the intestinal lesions and the oocysts were counted.The results showed that the booster immunization group was significantly different from the single immunization group in reducing intestinal lesions,increasing weight gain and anticoccidial index(p<0.05).The ACI of the single immunization group was mostly between 150 and 161,while the booster immunization groups was 160 and 181.Comprehensive comparison of ACI found that TEIN protein expressed by Pichia pastoris system was significantly different from E.coli(p<0.05).After the booster immunization,the recombinant TEIN protein expressed by Pichia pastoris was significantly different from the mixed group(p<0.05).The ACI of the recombinant TEIN protein expressed by Pichia pastoris against E.necatrix was up to 180.61,while the mixed protein was 160.60.Safety evaluation of tetravalent recombinant subunit vaccine for coccidia in chicken:The chickens were randomly divided into three groups including one-day-old single dose immunization group(100μg/feather),single dose three times immunization group(100μg/feather)and triple single dose immunization group(300μg/feather).The safety of the vaccine was comprehensively evaluated by observing the clinical symptoms,detecting blood routine and biochemical indexes,inspecting the pathological changes in the main internal organs.The results showed that there was no significant clinical response.There was no effect(p>0.05)on the blood routine and biochemical indexes.No obvious histopathological changes were found in the main organs after HE staining.which indicated that the vaccine was safe relatively.Evaluation on the immune duration and preservation of tetravalent recombinant subunit vaccine for coccidia in chicken:The immune duration was estimated by analyzing ACI,the titer of Ig G,IL-2,IL-10 and IFN-γin serum,counting the T spleen lymphocyte at 21d,35d and 49d after immunization.The storage time were evaluated by the physical properties,immune protection effect and safety stability for storing at 4℃,room temperature and-20℃respectively.The results showed that the ACI was 160.12 on the 49th day after immunization.The levels of Ig G,IL-2,IL-10 and IFN-γwere significantly higher than those of the control groups(p<0.05),the number of T lymphocytes was significantly increased(p<0.05).There was still a good immune protection effect when the vaccine was stored at 4℃for 180 days.In summary,The tandem TEIN gene was linked with GS Linker by E.tenella TA4,E.acervulina 3-1E,E.maxima IMP1 and E.necatrix NA4 was successfully expressed in Pichia pastoris and E.coli systems.On the basis,the tetravalent recombinant subunit vaccine of chicken coccidiosis was prepared.Through the immune protective test,it was found that the optimal immune dose was 100μg/feather.Comprehensive comparison of anticoccidial index showed that Pichia pastoris was better than E.coli expression system,the tetravalent recombinant protein was better than mixed protein immunization,enhanced immunization was better than single immunization.The results of safety evaluation showed that there were no abnormalities in blood indexes and main organs after immunization.The immune duration was at least 49 days,the vaccine still had good immune protection after 180days of storage at 4℃.which laid a good foundation for the clinical application of genetic engineering vaccine for chicken coccidiosis.