Lycium Barbarum Polysaccharide Promotes The Immunoprotective Effects Of Recombinant Lactobacillus Plantarum Vaccine Expressing Trichinella Spiralis Cathepsin F-like Protease 1 Gene

Trichinosis is a zoonotic parasitic disease that poses a serious health risk to humans.In most cases,it is caused by eating raw or undercooked meat products containing Trichinella spiralis encapsulations.T.spiralis has an unusual life cycle in which the adult,new larval,and muscle larval stages are all completed in the same host.Vaccines are currently an effective method of preventing Trichinosis,but they produce weak immunogenicity and low antibody levels.Vaccine combination adjuvant can effectively compensate for the low antibody level of vaccine alone in animals,so it is vital to develop effective adjuvant.It has been demonstrated that the polysaccharide used in traditional Chinese medicine is a type of low-toxic and biodegradable substance that is crucial in boosting vaccinations as adjuvants.T.spiralis cathepsin F(CPF)is a cysteine protease family papain subfamily member that hydrolyzes cysteine protease-specific substrates and participates in worm invasion.At the same time,CPF is the primary virulence factor in the T.spiralis infection process,and it is also the target of a number of anti-T.spiralis medication treatments.As a result,the recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene that we developed in our lab was used in this study.Simultaneously,using traditional Chinese medicine polysaccharides as a starting point,affordable and conveniently accessible traditional Chinese medicine Lycium barbarum polysaccharide was chosen as investigate their effects as immune adjuvants further.The following are the study’s key findings and contents:(1)To test the effect of Lycium barbarum polysaccharide on the immune enhancement of a recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene.Female BALB/c mice six weeks old were used as experimental animals.125 mice were randomly divided into five groups.The cells were divided into five groups:negative control(PBS),positive control(Ts),LBP,r Ts-CPF1,and r Ts-CPF1+LBP.The LBP group was given LBP(100mg/Kg/mouse)orally for 37 days.Each mouse in the r Ts-CPF1 group was given1.0×10~9CFU of live bacteria orally three times in a row,with a 10-day interval in between.The mice in the r Ts-CPF1+LBP group were immunized three times with recombinant Lactobacillus plantarum vaccine and given LBP orally for 37 days.PBS solution was administered orally to mice in the PBS and Ts groups for 37 days.The mice were then infected with 350 T.spiralis muscle larvae(T.spiralis ML),and flow cytometry was used to detect the expression of CD4~+IL-4~+and CD4~+IFN-γ~+in the Mesenteric Lymph Nodes(MLN)and Spleen.Indirect ELISA was used to detect specific Ig G,Ig G1,and Ig G2a antibodies in serum and specific s Ig A antibodies in intestinal lavage fluid.The m RNA transcription levels of IL-4 and IFN-γin the spleen were determined using RT-PCR.The reduction rate of adult intestinal worms and muscle larvae was assessed.To assess the pathological changes in the duodenum during the adult stage,as well as the tongue and masseter muscles during the muscle larval stage.The r Ts-CPF1 and r Ts-CPF1+LBP groups increased the expression of CD4~+IL-4~+and CD4~+IFN-γ~+in the MLN and Spleen,as well as the levels of specific Ig G,Ig G1 and Ig G2a in the serum and specific s Ig A in the intestinal lavage fluid.The expression of IFN-γand IL-4 was increased in the MLN and Spleen.The pathological changes in the duodenum,tongue,and masseter muscles of mice in the r Ts-CPF1 and r Ts-CPF1+LBP groups were improved after T.spiralis infection,with the r Ts-CPF1+LBP group having the best immunoprotective effect,with a reduction rate of 47.31%in adult worms and 68.88%in muscle larvae,respectively.LBP can boost T.spiralis vaccine immunoprotection and could be used as a new adjuvant for T.spiralis vaccine.(2)To test the effect of L-arabinose on the immune enhancement of a recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene.For various monosaccharides in Lycium barbarum polysaccharide,among which L-arabinose(L-Ara)is the main component,this study investigated whether L-Ara plays a significant role in immune enhancement.Female BALB/c mice aged 6 weeks were chosen as experimental animals.125 mice were randomly divided into five groups.They were the negative control(PBS),the positive control(Ts),the L-arabinose group(L-Ara)alone,the recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene group(r Ts-CPF1)alone,and the recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene combined with L-Ara group(r Ts-CPF1+L-Ara).For 37 days,the L-Ara group was given 150 mg L-Ara/day/mouse orally.Each mouse in the rTs-CPF1 group was given 1.0×10~9CFU live bacteria orally and immunized for three days in a row,with a 10-day interval.Mice in the r Ts-CPF1+L-Ara group were immunized three times with recombinant Lactobacillus plantarum vaccine and given L-Ara orally for 37 days.For 37 days,both the PBS and Ts groups were given the same volume of PBS solution orally.The experimental mice were then infected into 350 T.spiralis ML.In the same chapter,flow cytometry,RT-PCR,ELISA,and other related experiments were performed.The results showed that oral immunization with r Ts-CPF1 and r Ts-CPF1+L-Ara increased the expression of CD4~+IL-4~+and CD4~+IFN-γ~+in the MLN and Spleen of mice,as well as the level of specific Ig G in serum and specific s Ig A in the intestinal lavage solution.The pathological damage to the duodenum,tongue,and masseter muscle of mice in the r Ts-CPF1and r Ts-CPF1+L-Ara groups was improved after infection with T spiralis.The immune protection effect of the r Ts-CPF1+L-Ara group was the best,with adult and muscle larvae reduction rates of 32.19%and 36.58%,respectively.In this research,LBP and L-Ara were immunized with recombinant Lactobacillus plantarum vaccine expressing the T spiralis cathepsin F-like protease 1 gene.The results of the tests revealed that combining LBP and L-Ara with T spiralis vaccine can effectively increase the expression levels of CD4~+IL-4~+and CD4~+IFN-γ~+in the MLN and Spleen of mice,improve humoral and cellular immune responses,and provide good immune protection against T.spiralis infection.In conclusion,we discovered that LBP is a new Chinese medicine adjuvant for preventing T.spiralis infection,and this study offers a thought for the development of a novel anti-T.spiralis vaccine adjuvant.