| Tuberculosis(TB)was once a disease that was sentenced to death.Since 1940,with the discovery of anti-tuberculosis drugs and the continuous improvement of public health systems,the mortality rate has decreased.With the presence of drug-resistant bacteria,TB became a major threat to public health again.According to WHO's statistics in 2017,TB ravaged the world with nearly 1.5 million records each year and it already reached the top of all infected disease.TB is caused by Mycobacterium tuberculosis(Mtb),which is mainly transmitted through the respiratory tract.Mtb mainly infects the lungs causing pulmonary tuberculosis(PTB),and can also invade other tissues causing extrapulmonary tuberculosis.According to statistics,about 1.7billion people(5-10%)developed TB after infected with Mtb,but the morbidity is greatly increased among HIV patients.Risk factors for TB include malnutrition,diabetes,smoking,and alcohol abuse.The 2018 WHO tuberculosis report shows that90%of new cases are adults,with a male to female ratio of 2:1.TB mainly spreads in low-income developing countries(97%),and the annual cost of TB prevention,diagnosis and treatment is as high as 6.9 billion.Vaccination is the most cost-effective way to prevent TB.It can greatly control TB infection from the source and reduce the subsequent economic burden.This is of great significance to the development and improvement of public health.Bacille Calmette Guerin(BCG)is still the only TB vaccine available today with 80%global coverage.It provides protection for extrapulmonary tuberculosis in children,but does not protect against pulmonary tuberculosis of any age.WHO's 2030 goal:Compared to 2015reducing TB mortality by 90%and morbidity by 80%.To achieve this goal,the current BCG vaccine and prevention methods alone will not work.Although researchers have overcome the difficulties in vaccine development,a small number of candidate vaccines have entered the clinical trial stage.However,there is still no vaccine candidate that can replace BCG,and with the increasingly severe global TB prevention and control situation,it is urgent to break the routine and is imperative to introduce new technologies and new ideas to develop new TB vaccines.In recent years,more and more studies found that the IVT mRNA vaccine has become a rising star in the vaccine and has played an important role in the treatment of tumors and protein replacement therapy.IVT mRNA synthesizes mRNA that encodes the protein of interest in a cell-free system in vitro.Once entering the body,it is captured by the body cells and presented on the cell surface,which activates the MHC class I-restricted CD8~+T cell response.At the same time,along with the discovery of the recombinant enzymes Chec9 gp60 and gp61,which can work in Mtb,the efficiency of constructing recombinant BCG(rBCG)by traditional homologous recombination method has been greatly improved.A new spring is coming!Based on the above status,this study explores new technologies for TB vaccine development from both new ideas and old traditions,broadens the path of TB vaccine research,and contributes to the development of subsequent TB vaccines.Therefore,this study is divided into two parts.In vitro synthesis and immunogenicity of Mycrobacterium tuberculosis Ag85B-mRNA vaccineOBJECTIVE:Tuberculosis is ravaging the whole world and new TB vaccines are urgently needed.In vitro transcription of Mycobacterium tuberculosis Ag85B-mRNA was carried out and its immunogenicity was evaluated.METHODS:The Ag85B coding sequence was optimized by combining codon preference,GC content and free energy of mRNA secondary structure,and the?globin3'and 5'UTR sequences were inserted at both ends.The sequence of interest is synthesized and subjected to in vitro transcription.It was identified by agarose gel electrophoresis.After transfection into cells and expression in vitro was confirmed,the Ag85B-mRNA were inoculated into BALB/c mice with protamine to stimulate specific cellular immune responses.RESULTS:Secondary structure analysis of Ag85B optimized sequence shows higher stability.The Ag85B-mRNA were stable in vitro.Western Blot showed after Ag85B-mRNA transfection into HEK 293T cells for 24 and 48 hours,there were clear and specific target protein bands.The results of mouse immunoassay showed that Ag85B-mRNA could induce high levels of IFN-?against mycobacterial Ag85B that was responsible for Th1-type immune response.CONCLUSION:The synthesis of stable Ag85B-mRNA vaccine in vitro has good immunogenicity,which lays a foundation for the improvement of mRNA vaccine research and provides new ideas for the development of tuberculosis vaccine.Study on the construction of a novel recombinant BCG vaccine using homologous recombination systemOBJECTIVE:Mtb changes macrophages as a“base”to escape from immune surveillance and immune response through inhibiting phagocytosis-lysosomal maturation.Previous studies showed that urease is a virulence factor for a variety of pathogenic agents.Nitrogen produced by urease in Mtb is considered to be an important source of alkalized Mtb survival microenvironment,which not only prevents the acidification of phagosomes,but also prevents the fusion of phagosomes and lysosomes.Therefore,this part aims to screen out the ureC deletion BCG strain,and establish a rapid and efficient homologous recombination technology platform,and subsequently add various antigens of Mtb(metabolic active or dormant period)to this recombinant strain,and more important to lay the foundation of the new rBCG vaccine.METHODS:First,electroporating the plasmid pSL002 which expresses gp60 and gp61 recombinase into BCG.Both left and right homologous arms were constructed on plasmidpSL001,whichcontainsloxPfragment,-lefthomologous arm-loxP-hyg-GFP-loxP-right homologous arm-.The DNA fragment was electroporated into BCG containing plasmid pSL002 to obtain a double-exchanged ureC deletion strain,and then the plasmid pSL003 expressing Cre recombinase was electrotransferred into a successfully double-exchanged mutant,and the resistance gene and GFP was sheared.Since both plasmids pSL002 and pSL003 contain the SacB gene,plasmids pSL002 and pSL003 were finally removed using a 2%sucrose plate.RESULTS:The left and right homologous arms were successfully constructed,and the fragment of-left arm of ureC-loxP-hyg-GFP-loxP-right arm of ureC-was obtained.The sequencing results showed that it was completely correct.The electroporation of plasmid pSL002 is in progress.CONCLUSION:The long cycle of BCG growth is about 21 days.The ureC deletion strain is to inhibit the ability of Mtb to block the phagocytic lysosome maturation,so macrophages can fully digest and decompose their antigen into short peptides,which are presented on the cell surface for identification by immune cells.Furthermore,it is also possible to add various antigens(metabolic active phase or dormant phase)of Mtb to the ureC-deficient strain,which could not only prevent the onset of TB,but also prevent latent TB,and lays a foundation for constructing a novel recombinant BCG vaccine. |
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