| Trichinella spiralis is a dioecious nematode,can affect all kinds of mammals(including humans),some predatory birds and reptile.Pig is one of the most important reservoir host species for Trichinella spiralis.A large proportion of reports of people infected with Trichinella spiralis is due to patients who had raw or not fully cooked pork products infected with T.spiralis muscle larva.Therefore,it is of great significance to prevent the pigs from infection with Trichinella spiralis.Early experiments in our lab had found several strong immunorectivity antigen genes in cDNA library of different life stage of T.spiralis with infected pig serum(26dpi and 60dpi).Immune protective in mice model of some of these antigens had been completed.In this study,on the base of the results of the early experiments,we choosed 5 antigen genes(Ts-clp,Ts-zh68,Ts-T668,Ts-serpin and Ts-p45),whith had shown some extent protective against T.spiralis in mouse model and also suspected to be important functional genes involved in parasite survive,parasitic or immune escape.The antigen genes of Ts-p43 and Ts-p53 were also research in this study,for the reason that they had shown high protective immune response against T.spiralis infection in mice model.In this study,Changbai pigs were used as the experiment animal model,the protective immune response of above-mentioned seven antigen genes were studied.This study will help to provied high efficient candidate antigens for Trichinosis vaccine and provide valuable datum.HIS labeled recombinant proteins of the above seven antigens were expressed by using e.coli prokaryotic expression system,then these recombinant proteins were purified by nickel affinity chromatography column and followed with purpose strip recycling from SDS-PAGE electrophoresis on a large vertical plate.The target proteins in the colloidal particles were dissolved out in PBS solution,and then concentrated by ultrafiltration.Finally,high purity of target proteins were confirmed by SDS-PAGE electrophoresis.The immunorectivity of the target proteins were confirmed by western blot analysis with T.spiralis infected pig serum(60 dpi).It is critical of adjuvant screening for research and development of the recombinant protein vaccine.Freund’s adjuvant is the most widely used adjuvant in study,but due to its toxicity and negative effects on meat quality,its clinical application in animal vaccines was limited.Some new adjuvants appeared good in vaccines for trichinella infection in mice model,but given the diversity of the antigen used in these studies,it is unable to determine the best adjuvants.Therefore,in this study,rTs-serpin was used as a representative for recombinant antigens,formulated with three different types of adjuvants(Montanide ISA201,Montanide 1313 N IMS PR VG and freund’s complete and incomplete adjuvant),and the protective immune response of these formulation were evaluated in the T.spiralis infected mice model.In particular,the ability of promoting the humoral immune response and cellular immune response were evaluated.The results shown that compared with other two kinds of adjuvants formulations,IMS1313 adjuvants added group induced the best and most enduring IgG antibody and two weeks aheaded the highest peak of IgM.Additionally,high levels of Th1 and Th2 cytokines in serum were induced in IMS1313 adjuvant add group.In a word,Montanide IMS 1313 N PR VG,which is a liquid nanoparticles adjuvants combined with water dispersed immune stimulation complex,shown a quite protective with freund’s adjuvant and a better immune enhancement effect than that of freund’s adjuvant.Montanide 1313 N IMS PR VG is an ideal adjuvant for trichinella vaccine research.And this adjuvant also has the advantages of no need of emulsification and easy to use.Therefore,this adjuvant was selected for the subsequent research.Changbai pigs were used as experimental animals to assess the protective against T.spiralis infection of above seven recombinant proteins.Sixty piglets were divided into 10 groups,including of seven recombinant protein groups,a PBS group and a PBS + IMS1313 adjuvant group,and a blank control group,with six two months old piglets in each group.Twice immunization with 4 weeks interval were conducted,1 mg recombinant protein was injected to each pig for each vaccination.3 weeks after the last vaccination,each pig was oral infection with 5000 T.spiralis muscle larvae(except the pigs in the blank control group).Six weeks after trichinella infection,all pigs were sacrificed,the muscle larvae density(average LPG)of each group was determined for 15 different anatomic sites.For each anatomic site,up to 50 g and no less than 5 g of tissue was collected.The muscle larvae reduction rate of each group were calculated and compared with the PBS control group.The results shown that the seven recombinant proteins appeared large different in protective against T.spiralis infection in pigs model from their appearance in mice model.The muscle larva reduction rate of rTs – T668,rTs-clp,rTs-zh68,rTs-serpin,rTs-p45,rTs-p43 and rTs-p53 in pigs infection models were 51.31%,31.03%,45.21%,31.03%,3.66%,38.92% and 2.39%,respectively.Two recombinant proteins,which shown a relatively higher muscle larva reduction rate(more than 40%)derived from adult stage and newborn larvae stage of trichinella,both belong to the early life stage of trichinella.It means that the key protein molecules in early life stage of trichinella spiralis can serve as good candidate antigens for trichinella vaccine research.Additionally,the diaphragm of each pig was collected,fixed by formaldehyde,embedded in paraffin,sliced up.Then after hematoxylin-eosin staining,morphological of trichinella cyst in diaphragm of different groups were observed and compared.The results shown that the trichinella cyst appear morphological changes for different degree in all recombinant proteins immunization group.And the strength of the inflammatory cells invasion around the cyst is also different in different groups.However,trichinella cyst no obvious difference had been found in PBS + IMS1313 group compared to PBS group.More rounded trichinella cyst have been observed in rTs-T668 and rTs-clp group,but collagen layer structure have no obvious changes relative to other groups.Trichinella cyst in rTs-zh68,rTs-p45,rTs-p53 and rTs-p43 groups appeared edge irregular or collagen layer thinning in different degrees.And no obvious change in rTs-serpin group in trichinella cyst.Furthermore,inflammatory cells invasion,surrounding the cyst,in rTs-zh68,rTs-T668 and rTs-p43 groups are relative severe than other groups,it suggested that more serious damage of collagen capsule in these group.However,only mild inflammatory cells invasion were observed in rTs-clp,rTs-serpin,rTs-p45 and rTs-p53 groups.In order to further understand the immune protective effect of recombinant proteins,and evaluate their ability of inducing protective immune response against trichinella spiralis infection.The humoral immunity and cellular immune response after vaccination were assessed,specific IgG,IgG1,IgG2 and IgM antibody levels change trend,Th1(IFN-gamma,IL-2)and Th2 type(IL-10,IL-4)cytokines level change trend in serum were tested.Furthermore,the changes in T lymphocyte and B lymphocyte at two weeks after the last vaccination,the changes in natural immune cells(macrophages and neutrophils)at 6dpi were tested.Results show that Th1 dominanted Th1 / Th2 mix immune response were induced in rTs-T668,rTs – clp and rTs-zh68 group for all the time or a short period after vaccination.Th1(IFN-gamma,IL-2)and Th2 type(IL-10,IL-4)cytokine levels were significantly elevated(p<0.05)in all recombinant protein vaccinated groups two weeks after the last vaccination.Compared with the PBS control group,the level of macrophages were significantly(p<0.05)elevated in rTs – clp and rTs-p43 groups,it means that the immunosuppressive action to macrophages induced by T.spiralis infection have been eliminate in this two recombinant protein vaccinated groups.Compared with the blank control group,which was not infected with T.spiralis,the level of neutrophils dropped significantly in PBS(p<0.05),adjuvant + PBS group,rTs-p45 and rTs-p53 groups at 6dpi,suggesting that significant immunosuppressive action to neutrophils has been induced by T.spiralis infection.However,the level of neutrophils slightly rised,though not significant,in rTs-clp and rTs-p43 groups at 6dpi,it means that immunosuppressive action to neutrophils induced by T.spiralis infection has been eliminated.Comprehensive the results above,we found that the protective effect against trichinella in pigs induced by recombinant proteins may be related to several protective immune response factors.Induction of type Th1 dominanted Th1 / Th2 mix immune response and promoting to maintain normal levels of natural immune response may be the key in inducing of protective immune response agaist trichinella infection in pigs. |
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