| Corona Virus Disease 2019(COVID-19)is a violent zoonotic disease caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).It is highly infectious and has a high mortality rate.Humans,minks,dogs and cats are all susceptible.On March 11,2020,the World Health Organization(WHO)announced that COVID-19 has entered a global pandemic,becoming the most serious "public health event of international concern" and "major crisis once in a century" in 100 years.As of March 9,2021,the new coronavirus pneumonia has spread to 220 countries and regions,with a total of 117,647,370 confirmed cases and 2,607,383 deaths,posing a huge threat to global public health security.At present,the intermediate animal host of SARS-CoV-2 is still unclear.Although studies have shown that dogs and cats can carry SARS-CoV-2,it is not clear whether companion animals with poison will transmit SARS-CoV-2 to humans.Therefore,it is necessary to develop a safe and effective SARS-CoV-2 veterinary vaccines to prevent companion animals(cats,dogs)and special economic animals(mink)from being infected with SARS-CoV-2.It is of great significance to reduce the potential risk of SARS-CoV-2 transmission from animals to humans.Currently,there are 204 COVID-19 vaccines in the development stage,80 of which have entered human clinical trials,and more than 10 vaccines have entered phase III clinical trials.Inactivated vaccines,m RNA vaccines,adenovirus vector vaccines and subunit vaccines are at the forefront of vaccine research.Three inactivated vaccines,two m RNA and one adenovirus vector vaccines have been approved for marketing at home and abroad.However,there are relatively few studies on veterinary COVID-19 vaccines.In this paper,we prepared bacterial-like particle vaccines(BLPs)against the receptor binding domain(RBD)protein of the spike protein(S)of SARS-CoV-2.And conducted preliminary studies on its immunogenicity.The specific research content is as follows:1.Prokaryotic expression and purification of SARS-CoV-2 RBD proteinThe SARS-CoV-2 RBD gene was cloned into the prokaryotic expression vector pET30a(+),the recombinant expression plasmid pET30a(+)-RBD was constructed and transformed into the BL21(DE3)host bacteria to induce the expression of the target protein,and the expression conditions(expression time,expression Stabilization and inducer concentration)after optimization,use His-Ni+ column to purify the target protein.The results of the study showed that the target protein exists in the form of inclusion bodies,with a molecular mass of about32 KDa,and the highest expression level is under the conditions of a final concentration of IPTG of 0.6 mmol/L and induction at 37°C for 5 hours.As determined by the BCA method,18 mg of high-purity target protein can be purified per liter of recombinant bacterial solution after induced expression.Western blot results show that the target protein can be recognized by the rabbit polyclonal antibody and produce specific reaction bands,indicating that the recombinant protein has good reactogenicity.This will lay the foundation for the establishment of a method for the subsequent detection of specific SARS-CoV-2 antibodies in this study.2.Establishment of an indirect ELISA method for detection of SARS-CoV-2 antibodyUsing purified SARS-CoV-2 RBD protein as the coating antigen,high immune horse serum as the primary antibody,and HRP-labeled rabbit anti-horse IgG as the secondary antibody,an indirect ELISA for the detection of SARS-CoV-2 RBD specific IgG antibodies was established.Optimal detection conditions: antigen coating concentration of 1μg/m L,37℃ blocking for 1.5h,serum to be tested at 37℃ for 2h,and secondary antibody dilution of 1:2000.The specificity of the indirect ELISA test method was tested,and the results showed that the test method is compatible with the positive sera of severe acute respiratory syndrome coronavirus type 1(SARSCoV-1),Middle East respiratory syndrome coronavirus(MERS-CoV),and swine epidemics.The positive sera of PEDV,feline infectious peritonitis virus(FCoV),Marburg virus(MARV),Ebola virus(EBOV),and Rift Valley fever virus(RVFV).There is no cross-reaction and good repeatability,indicating that the indirect ELISA detection method has good specificity.This laid the foundation for the follow-up SARS-CoV-2 serological test.3.Construction and identification of SARS-CoV-2 bacteria-like particlesFirstly,the insect cell-baculovirus expression system was used to construct and rescue the recombinant baculovirus r BV-RBD-linker-PA3 with the SARS-CoV-2 RBD fragment,and after the identification was correct,the fusion protein RBD-linker-PA3 was expressed in batches.Secondly,MG1363 Lactococcus lactis was used to prepare GEM particles and combined with the fusion protein RBD-linker-PA3 to form bacteria-like particles GEM-RLP3.Genomic PCR identification showed that the recombinant baculovirus was successfully constructed.Indirect immunofluorescence and Western Blot results showed that the fusion protein RBD-linker-PA3 was successfully expressed and the protein was soluble expression.The results of indirect immunofluorescence,SDS-PAGE and Western Blot showed that the fusion protein was successfully displayed on the surface of GEM particles with good specificity.The results of SDSPAGE and gray-scale analysis showed that the amount of anchor binding between the fusion protein and GEM particles was 208.3 μg/U.Frozen section analysis of Lactococcus lactis and GEM particles bound to the fusion protein before and after treatment showed that both GEM particles and GEM-RLP3 bacteria-like particles were successfully prepared,which provided immunity for the experimental immunization study of bacterial-like particle vaccines in this study.original.4.Experimental immune study of SARS-CoV-2 bacteria-like particlesGEM-RLP3 particles were mixed with Freund’s adjuvant to prepare SARS-CoV-2 bacteriallike particle vaccine to immunize mice.The adjuvant alone group and the PBS group were used as controls to initially evaluate the immunogenicity of the vaccine.Neutralizing antibodies can be detected after the first immunization,but only slightly higher than the control group;the average serum neutralizing antibody titers for the first and second booster immunizations are 1:426 and1:597.After three immunizations,the detection results of specific IgG antibodies in mouse serum were 1:2048,1:32768 and 1:65536.The results of ELISpot detection,splenic lymphocyte proliferation level and cytokine secretion level showed that the vaccine can effectively promote the proliferation of splenic lymphocytes and the expression of Th1 and Th2 related cytokines after immunizing mice.It shows that the SARS-CoV-2 bacterial-like particle vaccine has good immunogenicity in mice and can induce the body to produce humoral and cellular immune responses.In summary,the SARS-CoV-2 bacterial-like particles prepared in this study have a good immune effect and can stimulate the body to produce specific cellular and humoral immune responses,laying a foundation for the development of a new SARS-CoV-2 veterinary vaccine Early-stage foundation. |
