CN116262794A - Recombinant mycobacterium tuberculosis antigen, preparation method and application thereof - Google Patents
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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Abstract
The invention provides a recombinant mycobacterium tuberculosis antigen, a preparation method and application thereof. The recombinant mycobacterium tuberculosis antigen can effectively induce immune response and has obvious protective effect on mycobacterium tuberculosis infection.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a recombinant mycobacterium tuberculosis antigen, a preparation method and application thereof.
Background
Tuberculosis (TB) is an important infectious disease that threatens human health. According to the global tuberculosis report issued by the world health organization 2020, 1000 tens of thousands (ranging from 890 to 1100 tens of thousands) of people are estimated to suffer from tuberculosis in 2019, and this number has been reduced at a very slow rate in recent years. The causative agent of tuberculosis is mycobacterium tuberculosis (Mycobacterium tuberculosis), which is transmitted mainly through the respiratory tract and can also enter the human body through the digestive tract. Mycobacterium tuberculosis infection can cause tuberculosis lesions of various systems or organs, such as tuberculosis, bone tuberculosis, renal tuberculosis, tuberculous meningitis, etc.
To date, BCG is the only vaccine approved worldwide to prevent tuberculosis, however its effect is controversial. Bcg is generally considered to be more effective in preventing severe tuberculosis, such as pediatric tuberculosis, tuberculosis meningitis, but is unable to prevent primary infection and has limited effect in preventing tuberculosis transmission. Other vaccines such as auxotrophic nuclear attenuated live vaccine, BCG recombinant vaccine, tuberculosis subunit vaccine, DNA vaccine and the like which have been developed have unsatisfactory protective effects, and no new vaccine can completely replace BCG at present.
Therefore, it is very necessary to develop a vaccine for preventing and treating tuberculosis which is more effective, safer and suitable for various people.
Disclosure of Invention
The invention aims to provide a recombinant mycobacterium tuberculosis antigen with a remarkable protective effect on mycobacterium tuberculosis infection.
In one aspect, the invention provides a recombinant Mycobacterium tuberculosis antigen comprising a fusion protein formed by at least two proteins or functionally active fragments thereof from PE35, PPE68, CFP-10 and Rv2627 c.
In some embodiments, PE35 comprises the amino acid sequence shown in SEQ ID NO. 1, PPE68 comprises the amino acid sequence shown in SEQ ID NO. 2, CFP-10 comprises the amino acid sequence shown in SEQ ID NO. 3, and Rv2627c comprises the amino acid sequence shown in SEQ ID NO. 4.
In some embodiments, the recombinant Mycobacterium tuberculosis antigen comprises a fusion protein formed by any two proteins or functional active fragments thereof in PE35, PPE68, CFP-10 and Rv2627c, the two proteins can be connected in any order, and the fusion protein formed by PE35 and PPE68 is exemplified by the fusion protein which can be PE35-PPE68 or PPE68-PE35.
In some embodiments, the recombinant Mycobacterium tuberculosis antigen comprises a fusion protein formed by any three proteins or functionally active fragments thereof of PE35, PPE68, CFP-10 and Rv2627c, which may be linked in any order, for example, a fusion protein formed by PE35, PPE68 and CFP-10, which may be PE35-PPE68-CFP-10, PE35-CFP-10-PPE68, PPE68-PE35-CFP-10, PPE68-CFP-10-PE35, CFP-10-PE35-PPE68 or CFP-10-PPE68-PE35.
In some embodiments, the recombinant Mycobacterium tuberculosis antigen comprises a fusion protein formed by four proteins or functionally active fragments thereof of PE35, PPE68, CFP-10 and Rv2627c, which may be linked in any order, e.g., the fusion protein may be PE35-PPE68-CFP-10-Rv2627c, PE35-CFP-10-PPE68-Rv2627c, PPE68-PE35-CFP-10-Rv2627c, PPE68-CFP-10-PE35-Rv2627c, CFP-10-PPE68-Rv2627c, rv2627c-CFP-10-PE35-PPE68 or Rv2627c-PPE68-PE35-CFP-10, etc.
In some embodiments, the recombinant mycobacterium tuberculosis antigen further comprises a polypeptide that enhances immunogenicity.
In some embodiments, the sequence of the immunogenicity enhancing polypeptide comprises an amino acid sequence shown in SEQ ID NO. 5.
In some embodiments, the recombinant Mycobacterium tuberculosis antigen comprises the amino acid sequence shown in any one of SEQ ID NO. 6-8 or any combination thereof.
In some embodiments, the recombinant Mycobacterium tuberculosis antigen further comprises the amino acid sequence shown in SEQ ID NO. 9.
In another aspect, the invention also provides a nucleotide sequence encoding the recombinant mycobacterium tuberculosis antigen.
In some embodiments, the nucleotide sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs 10-12.
In some embodiments, the nucleotide sequence further comprises the nucleotide sequence set forth in SEQ ID NO. 13.
In another aspect, the invention also provides a recombinant vector comprising the nucleotide sequence.
In another aspect, the invention also provides an expression system cell comprising the recombinant vector.
In some embodiments, the cells of the expression system comprise cells that are mammalian cells, insect cells, yeast cells, or bacterial cells, optionally; the mammalian cells include 293T cells or CHO cells and the bacterial cells include E.coli cells.
In another aspect, the present invention also provides a method for preparing the recombinant mycobacterium tuberculosis antigen, comprising the steps of:
constructing a recombinant expression plasmid by utilizing a nucleotide sequence for encoding the recombinant mycobacterium tuberculosis antigen;
transforming the constructed recombinant expression plasmid into host bacteria, screening the correct recombinant expression plasmid,
and transfecting cells of the expression system by using the screened recombinant expression plasmid, and collecting and purifying supernatant after expression to obtain the recombinant mycobacterium tuberculosis antigen.
In another aspect, the invention also provides application of the recombinant mycobacterium tuberculosis antigen, a nucleotide sequence for encoding the recombinant mycobacterium tuberculosis antigen, a recombinant vector containing the nucleotide sequence and an expression system cell containing the recombinant vector in preparation of tuberculosis vaccines.
In another aspect, the invention also provides a tuberculosis vaccine comprising the recombinant mycobacterium tuberculosis antigen and an adjuvant.
In some embodiments, the adjuvant is selected from one or more of an aluminum adjuvant, an MF59 adjuvant, an MPL adjuvant, a QS-21, GLA, cpG, AS01, an AS02, an AS03, an AS04 adjuvant.
The invention also provides a tuberculosis DNA vaccine, which comprises a DNA sequence for encoding the recombinant mycobacterium tuberculosis antigen.
In some embodiments, the DNA sequence comprises the sequence set forth in any one of SEQ ID NOs 14-16.
In some embodiments, the DNA sequence comprises the sequence set forth in SEQ ID NO. 17.
The invention also provides a tuberculosis mRNA vaccine comprising an mRNA sequence encoding the recombinant mycobacterium tuberculosis antigen.
In some embodiments, the mRNA sequence comprises the sequence set forth in any one of SEQ ID NOs 18-20.
In some embodiments, the mRNA sequence further comprises the sequence set forth in SEQ ID NO. 21.
The invention also provides a viral vector vaccine for preventing or treating tuberculosis, which contains a recombinant viral vector, wherein the recombinant viral vector contains a nucleotide sequence for encoding the recombinant mycobacterium tuberculosis antigen.
In some embodiments, the viral vector is selected from one or more of the following: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the invention provides a fusion protein formed by at least two proteins or functional active fragments thereof in PE35, PPE68, CFP-10 and Rv2627C, and experimental results show that fusion antigens such as P2P3, C4R7 and P2P3-C4R7 or combinations thereof can induce mice to generate obvious immune response, and have obvious protective effect on mycobacterium tuberculosis infection.
Drawings
The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention. Wherein:
FIG. 1 shows the expression of an antigen in BL21 (DE 3) according to the examples of the present invention;
FIG. 2 shows the expression of an antigen in BL21-Gold (DE 3) according to the examples of the present invention;
FIG. 3 shows CD4 production in 4 weeks after last immunization in the examples of the present invention + T cell cytokine detection results (×p)<0.05)；
FIG. 4 shows CD4 production 4 after 4 weeks of mice infected with Mycobacterium tuberculosis H37Rv in the example of the present invention + T cell cytokine detection results (×p)<0.05)。
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
In this application, the term "PE35" is a RD1 (region of difference 1) domain protein, 99 amino acids in total, as shown in SEQ ID NO:1, which interacts with PPE68, binds TLR2, stimulating macrophages to produce IL-10 and MCP-1.PE35 is soluble expressed in E.coli.
In this application, the term "PPE68" is a RD1 region protein of 368 amino acids in total, as shown in SEQ ID NO. 2, which is associated with the maintenance of infection by Bacillus tuberculosis, which interacts with PE35, and which also interacts with EspG1, eccA1, esxB in vitro, which also stimulates human production of IFN-gamma, and which polypeptide P9. It binds TLR2, stimulating macrophages to produce IL-10 and MCP-1.PPE68 is widely recognized by HLA-DR. PPE68 is soluble expressed in e.
In this application, the term "CFP-10" is a secreted protein, 100 amino acids in total, as shown in SEQ ID NO. 3, from which the initial Met is excised, and which elicits a strong T cell response. CFP-10 is expressed in E.coli in a soluble manner.
In this application, the term "Rv2627c" is a dormancy-associated antigen and a strong T-cell antigen, totaling 413 amino acids, as shown in SEQ ID NO. 4. IFN-gamma is induced in skin test positive human body, and expression is induced under low oxygen, low NO and low CO conditions. Rv2627c is soluble expressed in e.coli.
Example 1 amino acid sequence of Mycobacterium tuberculosis antigen
In the examples of the present invention, the present inventors devised a variety of Mycobacterium tuberculosis antigens, including P2P3 (PE 35-PPE 68), C4R7 (CFP-10-Rv 2627C) and P2P3-C4R7 (also referred to as 2347 in this application) fusion antigens.
The amino acid sequence of P2P3 is:
MEKMSHDPIAADIGTQVSDNALHGVTAGSTALTSVTGLVPAGADEVSAQAATAFTSEGIQLLASNASAQDQLHRAGEAVQDVARTYSQIDDGAAGVFAEMLWHAMPPELNTARLMAGAGPAPMLAAAAGWQTLSAALDAQAVELTARLNSLGEAWTGGGSDKALAAATPMVVWLQTASTQAKTRAMQATAQAAAYTQAMATTPSLPEIAANHITQAVLTATNFFGINTIPIALTEMDYFIRMWNQAALAMEVYQAETAVNTLFEKLEPMASILDPGASQSTTNPIFGMPSPGSSTPVGQLPPAATQTLGQLGEMSGPMQQLTQPLQQVTSLFSQVGGTGGGNPADEEAAQMGLLGTSPLSNHPLAGGSGPSAGAGLLRAESLPGAGGSLTRTPLMSQLIEKPVAPSVMPAAAAGSSATGGAAPVGAGAMGQGAQSGGSTRPGLVAPAPLAQEREEDDEDDWDEEDDW(SEQ ID NO:6)。
the amino acid sequence of C4R7 is:
MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGFMASSASDGTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDKLPIARPTIALAAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFYGNRRWLEKPSGFFAQPPPLTEVAVRKVKDRRRSFYRIFFDSGFTPHPGEPGSQRWLSYTANNREYALLLRHPEPRPWLVCVHGTEMGRAPLDLAVFRAWKLHDELGLNIVMPVLPMHGPRGQGLPKGAVFPGEDVLDDVHGTAQAVWDIRRLLSWIRSQEEESLIGLNGLSLGGYIASLVASLEEGLACAILGVPVADLIELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTPLVPMPGRFIYAGIADRLVHPREQVTRLWEHWGKPEIVWYPGGHTGFFQSRPVRRFVQAALEQSGLLDAPRTQRDRSA(SEQ ID NO:7)。
the amino acid sequence of the P2P3-C4R7 fusion antigen (2347) is:
MEKMSHDPIAADIGTQVSDNALHGVTAGSTALTSVTGLVPAGADEVSAQAATAFTSEGIQLLASNASAQDQLHRAGEAVQDVARTYSQIDDGAAGVFAEMLWHAMPPELNTARLMAGAGPAPMLAAAAGWQTLSAALDAQAVELTARLNSLGEAWTGGGSDKALAAATPMVVWLQTASTQAKTRAMQATAQAAAYTQAMATTPSLPEIAANHITQAVLTATNFFGINTIPIALTEMDYFIRMWNQAALAMEVYQAETAVNTLFEKLEPMASILDPGASQSTTNPIFGMPSPGSSTPVGQLPPAATQTLGQLGEMSGPMQQLTQPLQQVTSLFSQVGGTGGGNPADEEAAQMGLLGTSPLSNHPLAGGSGPSAGAGLLRAESLPGAGGSLTRTPLMSQLIEKPVAPSVMPAAAAGSSATGGAAPVGAGAMGQGAQSGGSTRPGLVAPAPLAQEREEDDEDDWDEEDDWAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAVVRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGFMASSASDGTHERSAFRLSPPVLSGAMGPFMHTGLYVAQSWRDYLGQQPDKLPIARPTIALAAQAFRDEIVLLGLKARRPVSNHRVFERISQEVAAGLEFYGNRRWLEKPSGFFAQPPPLTEVAVRKVKDRRRSFYRIFFDSGFTPHPGEPGSQRWLSYTANNREYALLLRHPEPRPWLVCVHGTEMGRAPLDLAVFRAWKLHDELGLNIVMPVLPMHGPRGQGLPKGAVFPGEDVLDDVHGTAQAVWDIRRLLSWIRSQEEESLIGLNGLSLGGYIASLVASLEEGLACAILGVPVADLIELLGRHCGLRHKDPRRHTVKMAEPIGRMISPLSLTPLVPMPGRFIYAGIADRLVHPREQVTRLWEHWGKPEIVWYPGGHTGFFQSRPVRRFVQAALEQSGLLDAPRTQRDRSAEDLVRAYHAMSSTHEQTVEDEARRMW(SEQ ID NO:8)
in addition, the mycobacterium tuberculosis antigen of the present invention may further comprise an M72 (MTB 39A (PPE 18) -MTB32A (PEPA) fusion protein, the amino acid sequence of which is:
MTAASDNFQLSQGGQGFAIPIGQAMAIAGQIRSGGGSPTVHIGPTAFLGLGVVDNNGNGARVQRVVGSAPAASLGISTGDVITAVDGAPINSATAMADALNGHHPGDVISVTWQTKSGGTRTGNVTLAEGPPAEFMVDFGALPPEINSARMYAGPGSASLVAAAQMWDSVASDLFSAASAFQSVVWGLTVGSWIGSSAGLMVAAASPYVAWMSVTAGQAELTAAQVRVAAAAYETAYGLTVPPPVIAENRAELMILIATNLLGQNTPAIAVNEAEYGEMWAQDAAAMFGYAAATATATATLLPFEEAPEMTSAGGLLEQAAAVEEASDTAAANQLMNNVPQALQQLAQPTQGTTPSSKLGGLWKTVSPHRSPISNMVSMANNHMSMTNSGVSMTNTLSSMLKGFAPAAAAQAVQTAAQNGVRAMSSLGSSLGSSGLGGGVAANLGRAASVGSLSVPQAWAAANQAVTPAARALPLTSLTSAAERGPGQMLGGLPVGQMGARAGGGLSGVLRVPPRPYVMPHSPAAGDIAPPALSQDRFADFPALPLDPSAMVAQVGPQVVNINTKLGYNNAVGAGTGIVIDPNGVVLTNNHVIAGATDINAFSVGSGQTYGVDVVGYDRTQDVAVLQLRGAGGLPSAAIGGGVAVGEPVVAMGNSGGQGGTPRAVPGRVVALGQTVQASDSLTGAEETLNGLIQFDAAIQPGDAGGPVVNGLGQVVGMNTAAS(SEQ ID NO:9)
the DNA sequence for encoding the antigen is codon optimized according to human as host to obtain the sequence SEQ ID NO. 14-17, which can be used for preparing DNA vaccine.
In the preparation of mRNA vaccines, the mRNA sequences of the antigens are SEQ ID NOs 18-21, respectively. The mRNA vaccine can be prepared by adding a 5' cap structure and a 5' UTR at the 5' end of the mRNA sequence and adding a 3' UTR and a polyadenylation sequence at the 3' end.
Example 2 construction of expression vectors
The DNA sequence for encoding the antigen is subjected to codon optimization according to an escherichia coli system to obtain a sequence SEQ ID NO:10-13, recombinant plasmids pRec5.0-P2P3, pRec5.0-C4R7, pRec5.0-2347 and pRec5.0-M72 are respectively constructed, and the recombinant plasmids are subjected to whole plasmid sequencing to ensure the sequence is correct.
Example 3 protein expression
1. Cell transformation
Competent cells BL21 (DE 3) and BL21-Gold (DE 3) pLsS were thawed on ice, and 2. Mu.l of plasmids pRec5.0-M72, pRec5.0-2347, pRec5.0-C4R7 and pRec5.0-P2P3 were added and gently mixed for 30min in an ice bath. And heat-shocking at 42 ℃ for 90s. Rapidly placing in ice bath for 2min, adding 1ml LB culture medium (preparation method: 10g peptone, 5g yeast powder, 10g NaCl, adding purified water to constant volume to 1L,121 deg.C, and sterilizing at 20 min), and resuscitating and culturing at 37deg.C and 200rpm for 30min. 100. Mu.l of the cultured bacterial liquid was plated on LB (kanamycin sulfate, kan, 100. Mu.g/ml, solarbio CAT: K1030) plates (preparation method: 10g peptone, 5g yeast powder, 10g NaCl,20 g agar powder, adding purified water to a volume of 1L,121 ℃, autoclaved for 20min, cooled to 50-55 ℃ C., and added with a resistant plating) and cultured overnight at 37 ℃.
2. Induction of expression
Single colonies on the plates were picked and inoculated into 5ml LB (Kan 100. Mu.g/ml) liquid medium and incubated at 37℃overnight at 200 rpm. Inoculating 300 μl of the cultured bacterial liquid into 30ml LB liquid medium (Kan 100 μg/ml), culturing for 2-3 hr, adding IPTG (Solarbio, CAT: I1020; final concentration 1 mM), inducing, culturing for 4-5 hr, and collecting bacteria.
20ml of post-induction bacterial liquid was taken, centrifuged at 4000rpm for 10min, the supernatant was discarded, and the pellet was resuspended in 2ml of Lysis buffer (containing 20mM Tris-HCl (pH 8.0), 150mM NaCl,20mM imidazole) and 50. Mu.l was taken as whole bacterial sample. 1ml of the heavy suspension was sonicated (total time 5min,10% power). After sonication, the mixture was centrifuged at 12000rpm for 4min at 4℃to take 50. Mu.l of the supernatant and 50. Mu.l of Lysis buffer as a sediment sample. The whole bacteria, supernatant and pellet samples were added with 50. Mu.l of 2×loading buffer (containing 50ml of 1M Tris (pH 6.8), 100ml of 1M DTT (Amresco CAT: 0281), 100ml of glycerol, 1g of bromophenol blue was weighed, mixed uniformly, and after complete dissolution, 200ml of 10% SDS was added, and water was added to a volume of 500 ml) and mixed uniformly, and SDS-PAGE was performed, and the results were shown in FIGS. 1 and 2.
The results show that: pRec5.0-2347/C4R7/M72 was expressed as inclusion bodies in BL21 (DE 3) and BL21-Gold (DE 3). pRec5.0-P2P3 was expressed in small amounts of soluble +inclusion bodies in BL21 (DE 3) and BL21-Gold (DE 3).
Example 4 protein purification
Cells were harvested by centrifugation of the induced bacterial liquid at 8,000g and 4℃for 15min, lysis buffer (50 mM Tris, pH8.0, 300mM NaCl) was added in a proportion of 5mL/g cells, incubated for 10min with stirring at room temperature, homogenized 3 times at 900bar, and the pellet was collected after centrifugation at 15000g and 4℃for 30min.
Washing buffer (50mM Tris,pH8.0,2M urea) was added at a ratio of 5mL/g cells to resuspend the pellet, the pellet was collected after centrifugation at 10000g and 4 ℃ for 15min, the procedure was repeated twice, and then the pellet was collected after centrifugation at 15000g and 4 ℃ for 15 min.
Denaturation buffer (50mM Tris,pH8.0, 200mM NaCl,8M urea) was added at a ratio of 5mL/g cells to resuspend the pellet and the supernatant was collected after centrifugation at 30000g and 4 ℃ for 30min.
Filtering the supernatant with 0.45 μm filter membrane, and performing affinity chromatography purification to obtain purified protein, wherein binding buffer (buffer A): 50mM Tris,pH8.0, 200mM NaCl,8M urea; elution buffer (buffer B) 50mM Tris,pH8.0, 200mM NaCl,8M Urea,500mM imidazole; loading flow rate: 3mL/min.
Example 5 mouse immunization experiment
Each antigen obtained in example 4 was diluted to 160. Mu.g/ml with antigen diluent (1 XPBS buffer) and mixed with AS01 adjuvant (liposomes containing 0.05mg/ml MPL and 0.05mg/ml QS 21) in equal volumes to produce a recombinant tuberculosis vaccine. A6-8 week female SPF grade C57BL/6N mouse model was used, with each mouse muscle injected with 0.1ml of recombinant tuberculosis vaccine (containing 8. Mu.g antigen) per time, with BCG (all responsible for the study of Chengdu Biotechnology, lot number 202004a 003) as positive control and physiological saline as negative control.
The invention also examines the combined action of M72 and other three antigens respectively, and the content of M72 and other three antigens is 4 mug (namely, the total content of antigens is 8 mug) when each injection is carried out.
The recombinant tuberculosis vaccine experimental group and the negative control group of the experiment adopt three immunization programs separated by 3 weeks, the unilateral hind limb is intramuscular injected, and the BCG immunization group adopts one immunization program and is subcutaneously administrated at the back. The immunization status of each group is shown in Table 1.
TABLE 1 mouse immunization status
Flow cytokine detection assays were performed on mice 4 weeks after the last immunization, and the remaining mice were given an aerosol infection with H37Rv strain at a dose of 100CFU. Cytokine detection, lung and spleen colony culture counts and pathological section detection were performed on mice 4 weeks after challenge, respectively, to evaluate immunogenicity of the recombinant tuberculosis vaccine in mice and protective efficacy against mycobacterium tuberculosis infection. In addition, during animal immunization, mice were weighed prior to injection on the day of immunization, and the state of the animals and the site of administration were observed after injection. During infection of animals, mice were observed for status on the day of infection, after which mice were monitored for body weight.
(1) Results of splenocyte detection in mice 4 weeks after last immunization
The 4 mice spleen cells in each group were subjected to protein stimulation and then stained for dead cells (FVS 450) and cell surface molecules CD3, CD4, CD8, CD154, and the intracellular cytokines IFN-gamma, IL-2 and TNF were stained after fixed rupture of membranes, and the positive proportion of each cytokine was analyzed separately. The results are shown in FIG. 3.
Overall, the specific cd4+ T cytokines produced after immunization with the four proteins of the invention were higher than the positive control BCG and the negative control. Among these four proteins, M72 protein produces minimal levels of specific CD4+ T cytokines (IL-2, IFN-gamma and TNF-alpha), and P2P3, C4R7, 2347 produce higher levels of cytokines than M72. Among the three proteins, the P2P3 protein immunity produces the lowest level of specific CD4+ T cell factor, the C4R7 protein immunity produces the higher level of specific CD4+ T cell factor than 2347 protein, and the M72 protein has no obvious improvement on the cellular immune response of the three antigens.
(2) Results of detection of spleen cytokines in mice 4 weeks after challenge
The 4-6 mice spleen cells in each group were subjected to protein stimulation, then stained for dead cells (FVS 450) and cell surface molecules CD3, CD4 and CD8, fixed membrane rupture was performed, and then stained for intracellular cytokines IFN-gamma, IL-2, TNF and CD154, and the positive proportion of each cytokine was analyzed respectively, and the detection results are shown in FIG. 4.
The results show that compared with the cytokine detection results 4 weeks after the last immunization, the cytokine level produced by the recombinant protein immunized mice 4 weeks after the challenge is significantly reduced. In addition to the 2347 protein, the other proteins produced higher levels of each cytokine than the positive control BCG, and the M72 group mice produced lower levels of each cytokine, and the M72+ C4R7 and M72+2347 group mice produced relatively higher levels of each cytokine. Mice in the 2347 group produced higher levels of IFN-gamma, while IL-2, TNF-alpha and CD154 levels were lower than the positive control BCG. The negative control (normal saline) mice produced elevated cytokine levels, possibly associated with immune responses following challenge.
(3) Mouse lung, spleen Colony Forming Unit (CFU) experiments
Mice 4 weeks after challenge were obtained from their left lung and spleen under sterile conditions, ground, serial diluted, plated with OADC-containing 7H10 plates, incubated at 37 ℃ for 4 weeks, and colony counts were performed to calculate the Colony Forming Units (CFU) of each mouse lung or spleen. The results showed that the lung and spleen Colony Forming Units (CFU) of the Negative Control (NC) mice were significantly higher than those of the positive control (BCG) mice, the lung and spleen Colony Forming Units (CFU) of each immunized group were significantly lower than those of the positive control (BCG) and Negative Control (NC), and that the lung and spleen Colony Forming Units (CFU) of each immunized group were not statistically different, wherein CFU of C4R7 and m72+c4r7 groups were relatively low, consistent with the results of the early cytokine detection, which indicated that the antigen of the present invention was effective in preventing mycobacterium tuberculosis infection.
(4) Pathological analysis of right lung tissue section of mouse
Right lung of each mouse is obtained aseptically, tissue paraffin embedding, slicing and the like are carried out through 4% paraformaldehyde fixation, hematoxylin and eosin staining is carried out on the tissue slice, and acid-fast staining is carried out. The protective effect of each group of mice on mycobacterium tuberculosis infection was assessed by scoring the lung lesions of the mice. The lung lesions scoring criteria are shown in table 2.
TABLE 2 pulmonary lesions scoring criteria
The lung lesion scoring result shows that the lung cell infiltration of the negative control group is obvious, the lung cell infiltration of the positive control group is moderate, and the lung of each experimental group of mice has only slight cell infiltration or very little inflammatory cell infiltration, but the lung lesions of the mice of each experimental group have no significant difference. The acid-fast staining results show that the lungs of the mice in the negative control group and the positive control group show obvious acid-fast staining positive mycobacterium tuberculosis thalli, while the lungs of the mice in each experimental group show only a small amount of acid-fast staining positive mycobacterium tuberculosis thalli, and the acid-fast staining results of the mice in each experimental group have no significant difference, wherein the lungs of the mice in the M72+C4R7 immune group have the least positive thalli, and the results further prove that the antigen in the invention can effectively prevent the mycobacterium tuberculosis infection.
(5) Weight change in mice
The mice weights were weighed and recorded before each dose, 28 days after the last dose, 14 days after challenge, 21 days, and 28 days, respectively. The results show that the weight of the mice is in a growing trend before the toxicity attack; the weight of the mice in the negative control group (Saline) after the toxicity attack shows an obvious continuous reduction trend, the weight of the mice in the positive control group also continuously reduces, but the reduction degree is lower than that of the mice in the negative control group, the weight of the mice in each protein immune group shows a slower reduction, and compared with the weight of the mice in the negative control group and the weight of the mice in the positive control group, the mice in each protein immune group have obvious differences, and the mice in each protein immune group have no obvious differences. The results show that the antigen in the invention can effectively reduce the damage of infection to mice.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (24)
1. A recombinant mycobacterium tuberculosis antigen comprising a fusion protein formed from at least two proteins or functionally active fragments thereof of PE35, PPE68, CFP-10 and Rv2627 c.
2. The recombinant mycobacterium tuberculosis antigen according to claim 1, wherein PE35 comprises the amino acid sequence shown in SEQ ID No. 1, PPE68 comprises the amino acid sequence shown in SEQ ID No. 2, CFP-10 comprises the amino acid sequence shown in SEQ ID No. 3, rv2627c comprises the amino acid sequence shown in SEQ ID No. 4.
3. The recombinant mycobacterium tuberculosis antigen according to claim 1 or 2, wherein the recombinant mycobacterium tuberculosis antigen further comprises a polypeptide that enhances immunogenicity.
4. The recombinant Mycobacterium tuberculosis antigen according to claim 3, wherein the sequence of the polypeptide for enhancing immunogenicity comprises an amino acid sequence shown in SEQ ID NO. 5.
5. The recombinant mycobacterium tuberculosis antigen according to claim 1, wherein the recombinant mycobacterium tuberculosis antigen comprises the amino acid sequence shown in any one of SEQ ID NOs 6-8 or any combination thereof.
6. The recombinant mycobacterium tuberculosis antigen according to claim 5, wherein the recombinant mycobacterium tuberculosis antigen further comprises the amino acid sequence shown in SEQ ID No. 9.
7. A nucleotide sequence encoding the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6.
8. The nucleotide sequence according to claim 7, wherein the nucleotide sequence comprises the nucleotide sequence set forth in any one of SEQ id nos 10 to 12.
9. The nucleotide sequence according to claim 8, further comprising the nucleotide sequence shown as SEQ ID No. 13.
10. A recombinant vector comprising the nucleotide sequence of any one of claims 7-9.
11. An expression system cell comprising the recombinant vector of claim 10.
12. The expression system cell of claim 11, wherein the cell of the expression system comprises a mammalian cell, an insect cell, a yeast cell, or a bacterial cell; alternatively, the mammalian cells comprise 293T cells or CHO cells and the bacterial cells comprise e.
13. A method for preparing the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6, comprising the steps of:
constructing a recombinant expression plasmid by utilizing a nucleotide sequence for encoding the recombinant mycobacterium tuberculosis antigen;
transforming the constructed recombinant expression plasmid into host bacteria, screening the correct recombinant expression plasmid,
and transfecting cells of an expression system by using the screened recombinant expression plasmid, and collecting and purifying supernatant after expression to obtain the recombinant mycobacterium tuberculosis antigen.
14. Use of the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6, the nucleotide sequence of any one of claims 7-9, the recombinant vector of claim 10, the expression system cell of claim 11 or 12 in the preparation of a tuberculosis vaccine.
15. A tuberculosis vaccine comprising the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6 and an adjuvant.
16. A tuberculosis vaccine AS claimed in claim 15 wherein the adjuvant is selected from one or more of aluminium adjuvants, MF59 adjuvants, MPL adjuvants, QS-21, GLA, cpG, AS01, AS02, AS03, AS04 adjuvants.
17. A tuberculosis DNA vaccine comprising a DNA sequence encoding the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6.
18. The DNA vaccine of claim 17, wherein the DNA sequence comprises the sequence set forth in any one of SEQ ID NOs 14-16.
19. The DNA vaccine of claim 18, wherein the DNA sequence further comprises the sequence set forth in SEQ ID No. 17.
20. A tuberculosis mRNA vaccine comprising an mRNA sequence encoding the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6.
21. The mRNA vaccine of claim 20, wherein the mRNA sequence comprises the sequence set forth in any one of SEQ ID NOs 18-20.
22. The mRNA vaccine of claim 21, wherein the mRNA sequence further comprises the sequence set forth in SEQ ID No. 21.
23. A viral vector vaccine for use in the prevention or treatment of tuberculosis, comprising a recombinant viral vector comprising a nucleotide sequence encoding the recombinant mycobacterium tuberculosis antigen of any one of claims 1-6.
24. The viral vector vaccine according to claim 23, characterized in that the viral vector is selected from one or several of the following: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors.
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