Evaluation Of The Immune Effect Of Lactobacillus Plantarum Expressing Novel Coronavirus S1 Antigen

Lactic acid bacteria play an important role in human and animal health because of their excellent probiotic function.They can not only colonize in the gut maintains the balance of the intestinal flora and also plays an important role in the regulation of the host’s immunity.Lactobacillus plantarum(Lb.plantarum)as a probiotic,it is often used in various antigen expression vectors and delivery vectors,and has been developed as a food grade expression host bacteria.Its advantage is that it can anchor foreign protein on the surface of host cells,reduce the hydrolysis of foreign protein and increase the local concentration of foreign protein,which is beneficial for antigen presenting cells to absorb foreign antigen and present it to T and B lymphocytes.Novel coronavirus pneumonia is a highly contagious disease caused by novel coronavirus(SARS-Co V-2),which has caused serious losses to people all over the world.Therefore,it is very important to develop a new vaccine to prevent virus infection.The spike protein of SARS-Co V-2 is divided into S1 and S2 subunits.The RBD domain of its C-terminal binds to ACE2 receptor in vivo,which mediates SARS-Co V-2 infection.At present,SARS-Co V-2 recombinant protein vaccine is mainly aimed at the development of its S protein.The key area of virus infection is RBD in S1 subunit.This suggests that both S protein and S1subunits can be considered to stimulate the body to produce immune response,so as to prevent novel coronavirus infection.Therefore,the S1 subunit of SARS-Co V-2 was selected as a foreign protein and anchored on the surface of Lactobacillus plantarum NC8 to evaluate the immune effect of recombinant Lactobacillus plantarum.This study is divided into four parts:(1)Construction and expression of recombinant protein S1 in lactic acid bacteriaThe sequence of novel coronavirus S1 protein gene was obtained from NCBI website.After optimization,the novel coronavirus S1 gene was synthesized,and then connected to p SIP409-pgs A’vector and transferred into DH5αcompetent cells.The results of restriction endonuclease digestion,PCR and sequencing showed that the recombinant plasmid p SIP409-pgs A’-S1 was successfully constructed.NC8-p SIP409-pgs A’-S1 recombinant Lactobacillus was successfully expressed and S1 protein was successfully anchored on the cell surface by immunofluorescence and Western blot.(2)Construction and purification of recombinant E.coli Expressing S1 ProteinThe S1 gene was cloned by PCR,ligated with p EASY Blunt Zero vector and transformed into DH5αcompetent cells.Then,the S1 gene fragment with sticky end was obtained by enzyme digestion.It was ligated into p ET-28a vector and transformed into BL21(DE3).The results of double enzyme digestion and sequencing showed that BL21-p ET-28a-S1 recombinant E.coli was constructed successfully.The expression of novel coronavirus S1 protein was verified by SDS-PAGE,and S1 protein was purified and purified,which laid the foundation for subsequent ELISA detection of specific antibodies and cell proliferation experiments.(3)Immune effect of NC8-p SIP409-pgs A’-S1 recombinant LactobacillusC57BL/6 mice were randomly divided into 5 groups with 8 mice in each group:saline group,NC8-p SIP409-pgs A’oral immunization group,NC8-p SIP409-pgs A’intranasal immunization group,NC8-p SIP409-pgs A’-S1 oral immunization group and NC8-p SIP409-pgs A’-S1 intranasal immunization group1.0×10~9 CFU per mouse.The first immunization was conducted on the 1st,2nd and 3rd day,and the booster immunization was conducted on the 15th,16th and 17th day.On the 25th day,the activation of dendritic cells(DC),specific cytokines in ileum and lung,spleen and mesenteric lymph nodes,specific s Ig A in feces and specific Ig G in serum were detected.The results showed that compared with the control group,CD80 and CD86 in DC of mouse Peyer’s lymph nodes were significantly activated(P<0.01);the detection of specific cytokines in mesenteric lymph nodes showed that compared with other control groups,the proportion of CD4~+IFN-γ~+cells in oral and nasal immunization group was significantly increased(P<0.05),and the proportion of CD8~+IFN-γ~+cells in oral recombinant Lactobacillus group was significantly increased(P<0.05)Compared with the control group,the proportion of CD4~+IFN-γ~+cells in the oral recombinant Lactobacillus group was significantly increased(P<0.05),the proportion of CD4~+IFN-γ~+cells in the nasal recombinant Lactobacillus group was significantly increased(P<0.05),and the proportion of CD4~+IFN-γ~+cells in the nasal recombinant Lactobacillus group was significantly increased(P<0.05)The proportion of CD8~+IFN-γ~+cells in acid bacteria group was significantly increased(P<0.05).These results indicate that the recombinant Lactobacillus group can effectively activate the DC surface of Paget’s lymph node,induce the cellular immune response of MLN and spleen in mice,and then activate T cells to regulate the immune response.Compared with the control group,the percentage of B220~+Ig A~+cells in Peyer’s lymph nodes of the oral and nasal recombinant Lactobacillus groups were significantly increased(P<0.05),and the number of B220~+Ig A~+cells in ileum and lung was significantly increased by immunofluorescence assay,The content of serum Ig G in the oral administration of recombinant Lactobacillus group was significantly increased on day 0 of booster immunization(P<0.05),and the content of serum Ig G in the oral administration and nasal drip of recombinant Lactobacillus group was significantly increased on day 7,14 and 21 of booster immunization(P<0.01).These results indicate that the recombinant Lactobacillus group can effectively activate B cells,promote the secretion of specific Ig G antibody,and then enhance the humoral immune level.In the detection of mucosal immune level of immunized mice,compared with the control group,the content of s Ig A in feces of mice in oral and nasal immunization groups increased significantly after 14 days of booster immunization(P<0.05);in the detection of ileal specific antibody SIg A,compared with other control groups,the content of ileal specific s Ig A in mice in oral recombinant Lactobacillus group increased significantly(P<0.05).The results showed that recombinant Lactobacillus could enhance intestinal mucosal immunity.