阅读说明：本技术 一种结核病候选疫苗融合蛋白 (Tuberculosis candidate vaccine fusion protein ) 是由 赵容川 鲍朗 于 2020-04-10 设计创作，主要内容包括：本发明涉及一种结核分枝杆菌ESAT6基因与RV1498a基因嵌合蛋白Dodecin-ESAT6候选疫苗的构建及其免疫原性研究,即利用基因工程技术将结核分枝杆菌ESAT6基因与RV1498a基因序列插入到同一大肠杆菌质粒pET28a的序列中,构建重组质粒rpET28a:RV1498a-ESAT6。然后采用热激转化的方法将上述载体导入大肠杆菌,表达融合蛋白Dodecin-ESAT6。本发明构建表达融合蛋白Dodecin-ESAT6,其免疫原性优于ESAT6。本发明提供了一种融合蛋白Dodecin-ESAT6的制备过程,并研究了其免疫原性,属于基因工程领域和结核疫苗领域。本发明将更有效的预防结核病的发生及传播。(The invention relates to construction of a mycobacterium tuberculosis ESAT6 gene and RV1498a gene chimeric protein Dodecin-ESAT6 candidate vaccine and immunogenicity research thereof, namely, a gene engineering technology is utilized to insert sequences of an ESAT6 gene and an RV1498a gene of mycobacterium tuberculosis into a sequence of a same escherichia coli plasmid pET28a to construct a recombinant plasmid rpET28a, RV1498a-ESAT 6. Then, the vector is introduced into escherichia coli by a heat shock transformation method to express the fusion protein Dodecin-ESAT 6. The expression fusion protein Dodecin-ESAT6 constructed by the invention has immunogenicity superior to that of ESAT 6. The invention provides a preparation process of a fusion protein Dodecin-ESAT6, researches the immunogenicity thereof, and belongs to the fields of genetic engineering and tuberculosis vaccines. The invention can effectively prevent the occurrence and the spread of tuberculosis.)

1. A recombinant protein Dodecin-ESAT6, wherein Dodecin is encoded by Mycobacterium tuberculosis Rv149 1498a gene and Esat6 is encoded by Mycobacterium tuberculosis RV3875 gene.

2. The recombinant protein Dodecin-ESAT6 of claim 1, which is more immunogenic than Esat 6.

3. The method for preparing the recombinant protein Dodecin-ESAT6 according to claim 1, wherein the method comprises the following steps:

(1) amplification of mycobacterium tuberculosis Esat6 and Rv1498a genes;

(2) amplification of the Rv1498a-Esat6 chimeric gene;

(3) inserting the Rv1498a-Esat6 chimeric gene into an Escherichia coli plasmid pet28a to construct rpET28a Rv1498a-Esat 6;

(4) the recombinant plasmid in (3) was heat-shocked into E.coli BL21(DE 3).

4. The recombinant protein Dodecin-ESAT6 of claim 1, wherein Dodecin-ESAT6 is obtained by Western-blot identification after antibiotic screening, PCR and induced expression.

5. A recombinant plasmid is characterized in that the gene sequences of Mycobacterium tuberculosis Rv1498a and Esat6 are inserted into the plasmid sequence of Escherichia coli pET28a after being embedded by using a genetic engineering technology.

6. The recombinant plasmid of claim 5 wherein the E.coli expression plasmid is pET28 a.

7. The recombinant plasmid of claim 5, wherein the chimeric gene sequence of Mycobacterium tuberculosis Rv1498a-Esat6 is inserted into the polyclonal site of the E.coli-Mycobacterium tuberculosis shuttle plasmid after ligation.

8. The recombinant plasmid of claim 5 wherein the recombinant plasmid rpET28a Rv1498a-Esat6 was obtained as identified by PCR and sequencing.

9. The recombinant protein Dodecin-ESAT6 according to claim 1, wherein the recombinant plasmid according to claim 5 is heat-shocked into BL21(DE3) to induce expression to obtain Dodecin-ESAT6 recombinant protein.

The technical field is as follows:

the invention relates to the field of genetic engineering and the field of novel tuberculosis vaccines, in particular to a fusion protein.

Background art:

tuberculosis (TB) is a chronic infectious disease seriously harming human health, and is caused by mycobacteria (mainly Mycobacterium tuberculosis), also called Tubercle Bacillus (TB), in recent years, the factors of population mobility, co-infection of Human Immunodeficiency Virus (HIV) and the tubercle bacillus, multiple drug resistance of the tubercle bacillus and the like provide new challenges for preventing and treating the tuberculosis.

The 6kD early secretory antigen (ESAT 6) of Mycobacterium tuberculosis is coded by RV3875 gene of RD (Regions of deletion, RD)1 region, which exists in the genome of a few pathogenic mycobacteria, is the region which is deleted firstly in the attenuation passage process of BCG, is related to virulence and antigenicity of Mycobacterium tuberculosis, and can be highly recognized by the immune system of a host. ESAT6 is a key virulence protein coded by the RD1 region, the virulence attenuation of the mycobacterium tuberculosis can be caused by the deletion or knockout of ESAT6 in the RD region, ESAT6 can induce peripheral blood mononuclear cells of a positive PPD skin test to generate proliferation reaction and IFN-gamma, then macrophages are activated, the growth inhibition effect and the killing capability of the macrophages on the intracellular tubercle bacillus are improved, and the protein is expected to become a main candidate molecule for developing a new vaccine. The gene coded by ESAT6 contains multiple T cell epitopes, and each site can induce T cell response. The B cell epitope of ESAT6 is located N-terminal to the polypeptide chain and its central region is identical to the central sequence of the T cell epitope. Due to the strong cellular immune activity and the unique coding gene sequence, the ESAT6 is expected to become a candidate gene of a novel vaccine. The results of Brandt et al, which injected mice subcutaneously with ESAT6 protein mixed with the adjuvants dimethyl dioctadecyl ammonium bromide (DDA) and monophosphoryl lipid a (mpl), showed that ESAT6 was effective as an effective ingredient in an anti-tuberculosis subunit vaccine. Olsen et al found that ESAT6 with DDA and MPL adjuvants had equivalent protective effects to BCG.

Dodecin protein is located near the lipL gene of the mycobacterium tuberculosis genome and is detected in a mycobacterium tuberculosis culture supernatant and a culture medium filtrate, research shows that the mycobacterium tuberculosis Dodecin protein is soluble secretory protein, the Dodecin protein is coded by the gene Rv1498a of the mycobacterium tuberculosis, and research shows that the Dodecin protein exists in a dodecamer form and can tolerate the temperature of 100 ℃ without changing the polymer structure of the Dodecin protein. The immunity of the body to tuberculosis is mainly completed through cellular immunity, the antigen which can cause immune response is usually large molecular protein, the immunogenicity of the protein with the molecular weight less than 10kd is extremely weak, and the antigen which is not added with the ESAT6 can only induce very weak cellular immunity and is probably related to the small molecular weight of the antigen. Dodecin protein and ESAT6 protein are combined into fusion protein, molecular weight is greatly improved, ESAT6 epitope is multiplied through polymer form, immunogenicity can be greatly improved, the defect that Esat6 protein immunogenicity is low is overcome, strong cell reaction is induced through the fusion protein form, the fusion protein is selected as molecular adjuvant to be recombined with mycobacterium tuberculosis immunity key antigen ESAT6, Dodecin-ESAT6 fusion protein which possibly has better immunogenicity and protective power through escherichia coli prokaryotic expression is taken as candidate vaccine, and great significance is provided for development of new tuberculosis vaccines, research of pathogenesis and prevention and treatment of tuberculosis in the future.

The invention content is as follows:

the invention aims to provide a tuberculosis candidate vaccine fusion protein and a preparation method thereof.

Primers are designed according to CDS sequences of ESAT6 and Rv1498a genes reported in Genbank, the ESAT6 and Rv1498a genes are respectively amplified, an Rv1498a-ESAT6 chimeric Gene is further amplified by an SOE method (overlap extension), the chimeric Gene and an Escherichia coli plasmid pet28a are simultaneously subjected to double enzyme digestion and inserted into a polyclonal site of the plasmid to construct a recombinant plasmid Rv1498a-ESAT6, a positive recon is screened by kanamycin, and then PCR and Gene sequence analysis are carried out for identification.

Taking out Escherichia coli BL21(DE3) competent bacteria from a refrigerator at the temperature of-80 ℃, placing the competent bacteria on ice for melting for three minutes, adding 10ul of the previously connected Rv1498a-ESAT6 plasmid, placing the plasmid on the ice for standing for 30 minutes, then quickly placing the plasmid into a water bath kettle at the temperature of 42 ℃ for heat shock of 85s, carrying out ice bath for 3min, adding a non-resistant LB liquid culture medium, and carrying out shake culture for 1 h. And uniformly blowing the bacterial liquid, dripping the bacterial liquid into an LB solid culture medium containing kanamycin resistance, uniformly coating, inverting the bacterial liquid into a bacterial culture box at 37 ℃, and culturing the bacterial liquid for the next day. Circular colonies were picked on solid plates. Adding the mixture into a bacterial lysate, centrifuging and taking the supernatant as colony PCR. The positive clones were expressed by induction with IPTG, lysed, and the lysate supernatant collected and purified with HIS trap affinity chromatography. SDS-PAGE and Western blot analysis show that Dodecin-ESAT6 fusion protein is obtained.

The candidate vaccine fusion protein has the following advantages:

1. recombinant expression of Mycobacterium tuberculosis key antigen ESAT 6.

2. The human Rv1498a gene and ESAT6 are fused together by genetic engineering technology.

3. Can stably express the foreign protein Dodecin-ESAT6 in the escherichia coli.

4. The immunogenicity of the recombinant vaccine is superior to that of ESAT 6.

Description of the drawings:

FIG. 1 is a PCR map of the Rv1498a gene, Esat6 gene and Rv1498a-ESAT6 chimeric gene. 1, DNA molecular weight standard; 2, Esat6 strip; 3, Rv1498a strip; 4, Rv1498a-ESAT6 band.

FIG. 2 shows the identification chart of rpET28a: Rv1498a-ESAT6 colony PCR. 1, DNA molecular weight standard; 2.3, rpET28a Rv1498a-ESAT6 recombinant Escherichia coli.

FIG. 3 is a plasmid map of pET28 a.

FIG. 4 shows the Dodecin-ESAT6 protein after SDS-page detection and purification. And 1, protein molecular weight standard. 2, purifying the Dodecin-ESAT6 protein.

FIG. 5 shows Dodecin-ESAT6 protein after Western-blot detection and purification. And 1, protein molecular weight standard. And 2, detecting the Dodecin-ESAT6 protein Western-blot after purification.

FIG. 6 levels of TNF-a, IL-12p40 in cell culture supernatants.

The specific implementation mode is as follows:

1. experiment main reagent

Culture medium reagent: tryptone (Tryptone), Yeast Extract (Yeast Extract) were purchased from Oxoid, UK;

gene cloning reagent: NdeI, Hind III restriction enzymes, T4DNA ligase were purchased from Thermo, Germany; dNTP, rTaq enzyme purchased from Takara; e.z.n.a.Plasmid Mini Kit I、E.Z.N.A.GelExtraction Kit is available from Omega, USA; the PCR primers were synthesized by Shanghai Biopsis corporation; the pet28a vector and the H37Rv strain genome are stored in a laboratory; BL21(DE3) is competent from Shanghai Biotech; RPMI1640 medium and fetal bovine serum were purchased from Gibco. Esat6 monoclonal antibody was purchased from Santa Cruz, USA; cytokine ELISA kits were purchased from boneuphratic. The endotoxin removal kit, ToxinEraser, was purchased from kasei corporation;

antibiotics: kanamycin (kanamycin) was purchased from Solarbio, beijing;

an inducer: isopropyl-beta-d-thiogalactoside (IPTG) was purchased from alatin, china;

2. experimental procedure

2.1PCR amplification

2 pairs of primers are designed according to CDS sequences of Rv1498a and Esat6 genes reported in Genbank, wherein the primer sequences are that the upstream of Rv1498a is 5'-GGAATTCCATatgagcaatcacacc-3', and the downstream of Rv1498a is 5-cgcctggaggattccga attcatgacagagcagcag-3', the upstream is the NdeI site, the downstream is the stop codon, and the underlined is the linker sequence containing 36 bases. 5' upstream of Esat6ctgctgctctgtcatgaattcggaatcctccagg cg5'-CCCAAGCTTctatgcgaacatccc-3' downstream of Esat6, 5'-CCCAAGCTTctatgcgaacatccc-3' upstream underlined sequence containing 36 bases complementary linker sequence, HindIII restriction site in bold type downstream genomic DNA of M.tuberculosis H37Rv as template, PCR amplification of Rv149 a gene and Esat6 gene separately, Rv1498a gene PCR amplification system with total volume of 50. mu.L, 5 × PCR Buffer 10. mu.L, dNTP 4. mu.L (dATP, dGTP, dCTP, dTTP 10mmoL/L), H37Rv genomic DNA 1. mu.L, Rv1498 gene upstream and downstream primers 1. mu.L, rTaq DNA polymerase 1U, deionized water 32.5. mu.L PCR conditions such as 95 ℃ pre-denaturation 5min, 94 ℃ denaturation 30 sec, 57 ℃ renaturation 45 sec, 72 ℃ extension 1min, 30 cycles, 72 ℃ extension 10 min, Esat 636 amplification method as above, the PCR amplification method using Rv1498 gene amplification template, Rsat 638 PCR amplification primer 1498 as template (Rva 3. 19. sup. V1498. 19. sup. 19. V. sup. 19. sup. RT).

2.2 enzyme digestion

HindIII and NdeI double enzyme digestion are respectively carried out on plasmid containing pET28a and Rv1498a-ESAT6 genes, and the enzyme digestion system and the reaction conditions are carried out according to the following table:

after adding, the mixture is fully and uniformly stirred by fingers, slightly centrifuged and placed in a constant-temperature water bath kettle at 37 ℃ for enzyme digestion for 2 hours. The resulting cleavage products were separated on agarose gel and used after cutting the gel using OMEGA e.z.n.a.The Gel Extraction Kit is recovered and purified, and the obtained product-20 is stored for later use.

2.3 ligation of the Gene of interest

The digested and purified pET28a is connected with a target gene Rv1498a-ESAT6 fragment by using the following connection system:

after the system is added, the mixture is gently vortexed and shaken, centrifuged and then placed in a PCR instrument for 16-degree connection for 4 hours, and the Dodecin-ESAT6 recombinant plasmid is obtained.

2.4 transformation of recombinant plasmids

Taking out Escherichia coli BL21(DE3) competent bacteria from a refrigerator at the temperature of-80 ℃, placing the competent bacteria on ice for melting for three minutes, adding 10ul of previously connected Dodecin-ESAT6 plasmid, gently bouncing uniformly, rapidly placing the competent bacteria on the ice for standing for 30min, rapidly placing the competent bacteria into a water bath kettle at the temperature of 42 ℃, thermally shocking for 85s, carrying out ice bath for 3min, adding 750ul of nonresistant LB liquid culture medium, placing the competent bacteria on a shaking table at the temperature of 37 ℃, and carrying out shaking culture for 1 h. Centrifuging the bacterium solution at 4500rpm for 5min, sucking 750 μ l of clear liquid, blowing the residual liquid uniformly, dripping into a solid culture medium containing 25 μ g/ml kanamycin LB, coating uniformly, placing in a bacterial incubator at 37 ℃, culturing for 12-16h times, and checking daily.

2.5 colony PCR Positive clones

Circular colonies with clear edges were picked on solid plates into 0.5ml EP tubes. 20ul of a bacterial lysate containing 10mM Tris-HCl pH8.0 and 0.1% Triton 100 was added and boiled in boiling water for 5 minutes. Centrifugation was carried out for 3 minutes at 10000 rpm, and 1ul of the supernatant was used for colony PCR with a pipette. The 10ul colony PCR system used was as follows:

after the PCR was completed, positive clones were identified by agarose gel electrophoresis (FIG. 2) and sequenced.

2.6 culture and inducible expression of bacteria

(1) The Dodecin-ESAT6 strain was removed from the-80 ℃ freezer, streaked on a kanamycin-resistant solid medium, and incubated at 37 ℃ in a constant temperature incubator for 16 hours.

(2) The next day, the LB plate was removed, and the round colonies were inoculated into 5ml of kanamycin-resistant liquid medium at 37 ℃ for 16h with shaking at 220 rpm.

(3) On the third day, 5ml of the bacterial liquid was taken out, inoculated into 300ml of a liquid medium with kanamycin resistance at 37 degrees, and subjected to shake culture for 1 hour at 220 ℃.

(4) Sampling at intervals to determine the OD600 value of the bacterial liquid, adding IPTG with the final concentration of 0.5-1mM to the culture medium when the OD600 value reaches 0.6, performing induction culture at 30 ℃ for 220 turns for 10 hours. During sampling, aseptic operation is performed to avoid contamination of bacteria liquid.

(5) And collecting the bacterial liquid after induction culture in a centrifuge tube, centrifuging for 1min by using a 4-degree centrifuge 13400 r, discarding supernatant, and repeatedly collecting all thalli.

2.7 ultrasonication extraction of Total protein

(1) The collected cells were resuspended in 20ml of Buffer A washing solution containing 20mM Tris-HCl pH7.4,1mM magnesium chloride.

(2) Centrifuge at 8000rpm for 2 minutes and discard the supernatant.

(3) The suspension was resuspended in 20ml of the above buffer.

(4) And (3) turning on the ultrasonic crusher, using an ultrasonic probe with the diameter of 5mm, adjusting the amplitude transformer to a corresponding gear, setting a program, performing ultrasonic treatment for 6s and stopping ultrasonic treatment for 6s at the power of 95% for 30 minutes.

(5) And opening the ultrasonic bin after a program is set, placing a centrifugal tube containing a large amount of bacteria liquid on ice, then placing the centrifugal tube into the ultrasonic bin for fixing, covering the bin door, and starting ultrasonic treatment. After the ultrasonic treatment is finished, the bacteria liquid is changed from turbid to clear, which indicates that the ultrasonic treatment is complete, and the ultrasonic treatment avoids the generation of a large amount of bubbles.

(6) And (4) centrifuging the bacteria liquid subjected to ultrasonic treatment for 2min at 13000rpm of a 4-degree centrifuge, collecting a supernatant protein solution, and storing the supernatant protein solution in a-20-degree refrigerator for later use.

2.8 purification of Dodecin-ESAT6 protein

(1) The 1ml Ni-NTA affinity chromatography column was taken out from the 4 degree refrigerator, the AKTA Primer purifier was turned on, a manual run was selected, the flow rate was set to 0.5ml/min, and the upper limit column pressure was set to 0.4 MPa.

(2) The Ni-NTA affinity chromatography column was mounted on the purifier to avoid air bubbles entering the column.

(3) The column was equilibrated with 10 volumes of equilibration buffer (20mM Tris-HCl pH7.4,0.5M NaCl,10mM imidazole) and the UV absorbance peak at OD280 was zeroed out without a drop.

(4) The total cleaved protein was loaded from loading port A. The flow rate was adjusted to 0.5 ml/min.

(5) After the loading is finished, the washing is continued by 10-20 times of the volume of the balance buffer solution until the OD280 absorbance value is not reduced any more.

(6) Gradient elution was performed using equilibration buffer containing 50, 100, 200mM imidazole, setting flow rate at 1 ml/min.

(7) The eluted Dodecin-ESAT6 protein was collected.

The collected Dodecin-ESAT6 protein was concentrated by ultrafiltration and subjected to SDS-PAGE (FIG. 4) and Western-blot (FIG. 5). Removing endotoxin by using an endotoxin removal kit, and storing in a refrigerator at-80 ℃.

2.9 Induction of cytokines

Dodecin-ESAT6 fusion protein, Esat6 protein and PBS are respectively added into THP-1 cell 24-well cell culture plates paved with 1E6cells/well, the plates are placed in a 5% CO2 cell incubator at 37 ℃ for 24h, cell culture supernatant is collected by centrifugation at 4000rpm, and secretion of TNF-a and IL-12p40 is detected by an ELISA kit (figure 6). The results show that: compared with Esat6, the Dodecin-ESAT6 fusion protein remarkably improves the secretion of human macrophage proinflammatory factor TNF-a (P < 0.001) and IL-12P40(P < 0.05).

Conclusion

The Dodecin-ESAT6 fusion protein is successfully expressed, the immunogenicity of the Dodecin-ESAT-6 fusion protein is superior to that of ESAT6, and a foundation is laid for developing a novel vaccine with specific anti-tubercle bacillus immunoprotection and adjuvant enhancement dual effects.

Sequence listing

<110> Sichuan university

<120> a tuberculosis candidate vaccine fusion protein

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<141>2020-04-10

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