CN103214582B - Immunogenic fusion protein for tuberculosis and application of immunogenic fusion protein - Google Patents

Abstract

The invention discloses an immunogenic fusion protein for tuberculosis and an application of the immunogenic fusion protein. The immunogenic fusion protein for tuberculosis, disclosed by the invention, is a fusion protein containing mycobacteria Ag85a and Rv2626c. The invention further discloses a recombinant attenuated listeria monocytogenes, and a genome of the recombinant attenuated listeria monocytogenes is integrated with an encoding gene of the fusion protein disclosed by the invention. The fusion protein or the encoding gene or the recombinant attenuated listeria monocytogenes disclosed by the invention can be used for preparing a tuberculosis vaccine.

Description

A fusion protein with immunogenicity against tuberculosis and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion protein with immunogenicity against tuberculosis and its application.

Background technique

Tuberculosis (TB) is a chronic infectious disease that seriously endangers people's health. At present, about 2 billion people are infected in the world. There are about 8-10 million new tuberculosis patients every year, and about 2-3 million deaths due to tuberculosis every year. At present, the number of tuberculosis patients in my country ranks second in the world, second only to India with an annual incidence of about 1.3 million. It is one of the 22 countries with severe tuberculosis epidemics in the world, and it is also one of the 27 countries with severe multi-drug-resistant tuberculosis epidemics in the world. , the annual death toll due to tuberculosis reaches 130,000, exceeding the sum of the death toll from other infectious diseases. Therefore, tuberculosis is one of the major diseases to be controlled in our country.

The pathogenic bacteria of tuberculosis are mainly Mycobacterium tuberculosis (hereinafter abbreviated as M.tb) and Mycobacterium bovis (hereinafter abbreviated as Mb) in the Mycobacterium genus. Mycobacterium is a typical type of intracellular parasitic bacteria. It can survive in the phagosome for a long time after being phagocytized by the host cell, and uses the phagosome as its own "safe haven", which greatly limits the body's immune response to it Ability. When the host is infected with mycobacteria, a small part of the host will directly form active tuberculosis, while most of the host will force the bacteria to enter a dormant state due to their own immune response, thereby controlling the further development of the disease. However, when the latently infected host is old or immunocompromised, the bacteria will reactivate and develop active tuberculosis. Currently, the only clinically available vaccine against tuberculosis is Bacillus Calmette-Guerin (BCG), and because BCG cannot provide protection for adults, most of the other newly developed tuberculosis vaccines are prophylactic vaccines based on the expression of antibodies in the early stage of tuberculosis infection , because these vaccines select antigens expressed in the early growth phase and logarithmic growth phase, they cannot induce the body to produce an immune response against latent antigens expressed in the dormant phase (quiescent growth phase or hypoxic state) of Mycobacterium tuberculosis. Latent infection is the main source of tuberculosis recurrence and one of the reasons why tuberculosis cannot be completely eliminated. It is imperative to develop new vaccines against these latent antigens.

According to studies in several literatures, the DosR (Dormancy Survival Regulator) regulatory system plays a vital role in the process of mycobacteria changing from an active state to a latent state. It can control the induced expression of 48 genes, and the expression of these genes is a necessary condition for mycobacteria to adapt and persist in the face of low oxygen, CO, NO and other conditions in the body. Rv2626c is the latency antigen encoded by DosR regulation. There is currently no tuberculosis vaccine prepared using Rv2626c.

Contents of the invention

The purpose of the present invention is to overcome the defects in the prior art and provide a fusion protein with tuberculosis immunogenicity and its application.

One aspect of the present invention provides a fusion protein, which is a fusion protein comprising mycobacterium Ag85a and Rv2626c.

In the fusion protein, the amino acid sequence of Ag85a is SEQ ID NO: 1:

FSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA。

In the fusion protein, the amino acid sequence of Rv2626c is SEQ ID NO: 2:

TTARDIMNAGVTCVGEHETLTAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS.

In the fusion protein, the Ag85a protein and the Rv2626c protein may optionally contain a connecting peptide.

The Ag85a protein can be connected to the N-terminal or C-terminal of the Rv2626c protein, preferably, connected to the N-terminal of the Rv2626c protein.

Further, the fusion protein also contains monocytogenes listeria hemolysin protein LLO.

Further, the fusion protein is a fusion protein of monocytogenes listeria hemolysin protein LLO and mycobacterium Ag85a and Rv2626c.

The amino acid sequence of the monocytogenes listeria hemolysin protein LLO is SEQ ID NO: 3:

KIMLVFITLILISLPIAQQTEAKDASAFNKENSISSMEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEINYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNSVDNPIE

In the fusion protein, the fusion protein of Ag85a protein and Rv2626c protein is inserted into the 37th and 38th amino acid residues of the aforementioned monocytogenes listeria hemolysin protein LLO (equivalent to the 37th amino acid residue of wild-type LLO). and amino acid residue 52).

Further, the amino acid sequence of the fusion protein is SEQ ID NO:4:

KIMLVFITLILISLPIAQQTEAKDASAFNKENSISSMFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDLPGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAFKAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYVAQFNISWDEINYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYPKYSNSVDNPIE

The fusion protein has strong immunogenicity against tuberculosis.

The second aspect of the present invention provides a polynucleotide encoding the fusion protein.

A polynucleotide of the invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded.

The third aspect of the present invention provides a vector containing the polynucleotide.

Methods well known to those skilled in the art can be used to construct the vector. These methods include recombinant DNA techniques, DNA synthesis techniques, and the like. The DNA encoding the fusion protein can be effectively connected to the multiple cloning site in the vector to guide mRNA synthesis and then express protein, or for homologous recombination.

Preferably, the vector is a prokaryotic vector or a shuttle plasmid, such as a prokaryotic vector pMD-20T, a shuttle plasmid pKSV7, and the like.

The fourth aspect of the present invention provides a host cell transformed with the vector.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; Salmonella typhimurium, Listeria; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS.293 cells, or Bowes melanoma Cells, animal cells, etc.

Among them, Listeria monocytogenes (Listeria monocytogenes, hereinafter abbreviated as LM), such as yzuLM4, is particularly preferred.

The five aspects of the present invention provide a recombinant attenuated Listeria monocytogenes, the recombinant attenuated Listeria monocytogenes genome is integrated with a fusion protein encoding mycobacterial Ag85a and Rv2626c gene, the recombinant attenuated Listeria monocytogenes bacteria can express the fusion protein of Listeria monocytogenes hemolysin protein LLO and mycobacterium Ag85a and Rv2626c.

Further, the gene encoding the fusion protein of mycobacterium Ag85a and Rv2626c is recombined and integrated between bases 111 and 154 of the hly gene in the genomic DNA of wild-type Listeria monocytogenes.

Further, in the fusion protein of the monocytogenetic Listeria hemolysin protein LLO and mycobacterium Ag85a and Rv2626c, the amino acid sequence of Ag85a is SEQ ID NO: 1, and the amino acid sequence of Rv2626c is SEQ ID NO: 2 , the amino acid sequence of monocytogenes listeria hemolysin protein LLO is SEQ ID NO:3.

In the fusion protein of the monocytogenetic listeria hemolysin protein LLO and mycobacterium Ag85a and Rv2626c, the monocytogenetic listeria hemolysin protein LLO, the Ag85a protein and/or the Rv2626c protein can be selected contain linker peptides.

Further, the amino acid sequence of the fusion protein of the mycobacterium Ag85a and Rv2626c is SEQ ID NO: 11:

FSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS。

Further, the nucleotide sequence of the gene encoding the fusion protein of mycobacterium Ag85a and Rv2626c is SEQ ID NO:5:

AAAAGGGCCGGCCCGAACGGCCACCTCATGGACGTCCACGGCAGCGGCAGCTACCCGGCACTGTAGTTCCAGGTTAAGGTTTCACCACCACGGTTGAGCGGGCGGGACATGGACGAGCTGCCGGACGCGCGCGTCCTGCTGAAGTCGCCGACCCTGTAGTTGTGGGGCCGCAAGCTCACCATGCTGGTCAGCCCGGACAGCCACCAGTACGGCCACCCACCGGTCAGTTCGAAGATGAGGCTGACCATGGTCGGGCGGACGCCGTTCCGGCCAACGGTCTGAATGTTCACCCTCTGGAAGGACTGGTCGCTCGACGGCCCCACCGACGTCCGGTTGTCCGTGCAGTTCGGGTGGCCTTCGCGGCAGCAGCCAGAAAGCTACCGACGAAGAAGCCGCGACTGCGACCGCTAGATAGTGGGGGTCGTCAAGCAGATGCGCCCTCGCTACAGCCCGGACAACCTGGGGAGGGTCCGCTACCCAGGGTGGGACTAGCCGGACCGCTACCCACTGCGACCGCCGATGTTCCGGAGGCTGTACACCCCGGGCTTCCTCCTGGGCCGCACCGTCGCGTTGCTGGGCGACAACTTGCAGCCCTTCGACTAGCGGTTGTTGTGGGCGCAGACCCACATGACGCCGTTGCCGTTCGGCAGCCTAGACCCACCGTTGTTGGACGGCCGGTTCAAGGAGCTCCCGAAGCACGCCTGGTCGTTGTAGTTCAAGGTTCTGCGGATGTTGCGGCCACCGCCGGTGTTGCCGCACAAGCTGAAGGGCCTGTCGCCATGCGTGTCGACCCTCATGACCCCCCGCGTCGAGTTGCGATACTTCGGGCTGGACGTTGCCCGTGACCCACGGTGCGGGTTGTGGCCCGGGCGCGGGGTCCCGCGGCCGCCACCGCCTAGGCCACCGCCACCGAGGTGGTGGCGTGCGCTGTAGTACTTGCGTCCACACTGGACACAACCGCTTGTGCTCTGCGATTGGCGACGGCGAGTTATGTACGCAC TCGTGCTGTAGCCGCGCAACGGCTAGACGCCCCTGCTGCTGGCCGACGTGCCGTACGAGTGGCTGGCGCTGTAACACTAGTTTCCGGACCGACGCCCGGATCTGGGCTTATGGCGGTGCCGACCGCTCAACCGGGCCCTGTCGTAGATGATGCAGCTACGCTTGCGTTCGTAGGTCCTCTACGAGTTGCAGTACCTTCTTGTAGTCCAGGCGGCACAAGGCCAGTAGAGTCTCGTGGCGAACCAGCCTTAGCAGTGGCTTCGGCTGTAGCGGGCTGTGGACGGGCTCGTGCGGTAACACGTCAAGCAGTTCCGTTAGACGAGCGGGTACCGGGAGCGGTCG

Further, compared with the wild-type Listeria monocytogenes, the present invention replaces bases 112-153 of the hly gene of the wild-type Listeria monocytogenes with SEQ ID NO:5. That is, in the recombinant attenuated Listeria monocytogenes of the present invention, the wild-type hly coding gene is replaced by the coding gene of the fusion protein whose sequence is SEQ ID NO:4.

The sixth aspect of the present invention discloses a method for constructing the recombinant attenuated Listeria monocytogenes, comprising the following steps:

1) Amplify the coding gene fragments of Ag85a (also known as Ag85A, FbpA, Rv3804c) and Rv2626c from the genome of Mycobacterium tuberculosis strain; The source arm fragment hlya and the downstream homology arm fragment hlyb;

2) The coding genes of Ag85a and Rv2626c obtained in step 1) and the upstream and downstream homology arm fragments were spliced into a hlya-Ag85a-Rv2626c-hlyb fusion fragment and ligated into a shuttle plasmid to obtain a recombinant shuttle plasmid;

3) The recombinant shuttle plasmid obtained in step 2) was transformed into Listeria monocytogenes, screened by double pressure of resistance and temperature, and then passaged without resistance to obtain recombinant attenuated monocytogenes monocytogenes Listeria bacteria.

Further, in step 1), the sequence of the amplified upstream homology arm fragment hlya is SEQ ID NO: 6:

AGGTTTGTTGTGTCAGGTAGAGCGGACATCCATTGTTTTGTAGTTACAGAGTTCTTTATTGGCTTATTCCAGTTATTAAGCGAATATGCTTTTCCGCCTAATGGGAAAGTAAAAAAGTATAAAATAAAACAGAGTAATAAAACTAATGTGCGTTGCAAATAATTCTTATACAAAATGGCCCCCTCCTTTGATTAGTATATTCCTATCTTAAAGTGACTTTTATGTTGAGGCATTAACATTTGTTAACGACGATAAAGGGACAGCAGGACTAGAATAAAGCTATAAAGCAAGCATATAATATTGCGTTTCATCTTTAGAAGCGAATTTCGCCAATATTATAATTATCAAAAGAGAGGGGTGGCAAACGGTATTTGGCATTATTAGGTTAAAAAATGTAGAAGGAGAGTGAAACCCATGAAAAAAATAATGCTAGTTTTTATTACACTTATATTAGTTAGTCTACCAATTGCGCAACAAACTGAAGCAAAGGATGCATCTGCATTCAATAAAGAAAATTCAATTTCA（525bp）

The sequence of the downstream homology arm fragment hlyb is SEQ ID NO: 7:

CCAATCGAAAAGAAACACGCGGATGAAATCGATAAGTATATACAAGGATTGGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGAAAAGGTTACAAAGATGGAAATGAATATATTGTTGTGGAGAAAAAGAAGAAATCCATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCAATTTCGAGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAATCAACCAGATGTTCTCCCTGTAAAACGTGATTCATTAACACTCAGCATTGATTTGCCAGGTATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCCACTAAATCAAACGTTAACAACGCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCAAATGTAAGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAATCACAATTAATTGCGAAATTTGGTACAGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGT（732bp）

The Ag85a gene is SEQ ID NO: 8:

TTTTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGGGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCC

The Rv2626c gene is SEQ ID NO: 9:

ACCACCGCACGCGACATCATGAACGCAGGTGTGACCTGTGTTGGCGAACACGAGACGCTAACCGCTGCCGCTCAATACATGCGTGAGCACGACATCGGCGCGTTGCCGATCTGCGGGGACGACGACCGGCTGCACGGCATGCTCACCGACCGCGACATTGTGATCAAAGGCCTGGCTGCGGGCCTAGACCCGAATACCGCCACGGCTGGCGAGTTGGCCCGGGACAGCATCTACTACGTCGATGCGAACGCAAGCATCCAGGAGATGCTCAACGTCATGGAAGAACATCAGGTCCGCCGTGTTCCGGTCATCTCAGAGCACCGCTTGGTCGGAATCGTCACCGAAGCCGACATCGCCCGACACCTGCCCGAGCACGCCATTGTGCAGTTCGTCAAGGCAATCTGCTCGCCCATGGCCCTCGCCAGC。

Further, in step 1), the Mycobacterium tuberculosis strain is the standard strain H37Rv. The Listeria monocytogenes strain described in steps 1) and 3) is the Listeria monocytogenes strain yzuLM4.

Further, step 2) can use overlapping derivation PCR technology (SOEing PCR) to splice each fragment, and then use restriction site to insert the spliced fragment into the shuttle vector.

Further, in step 2), the shuttle plasmid can be pKSV7.

Further, in step 2), the sequence of the spliced hlya-Ag85a-Rv2626c-hlyb fusion fragment is SEQ ID NO: 10:

AGGTTTGTTGTGTCAGGTAGAGCGGACATCCATTGTTTTGTAGTTACAGAGTTCTTTATTGGCTTATTCCAGTTATTAAGCGAATATGCTTTTCCGCCTAATGGGAAAGTAAAAAAGTATAAAATAAAACAGAGTAATAAAACTAATGTGCGTTGCAAATAATTCTTATACAAAATGGCCCCCTCCTTTGATTAGTATATTCCTATCTTAAAGTGACTTTTATGTTGAGGCATTAACATTTGTTAACGACGATAAAGGGACAGCAGGACTAGAATAAAGCTATAAAGCAAGCATATAATATTGCGTTTCATCTTTAGAAGCGAATTTCGCCAATATTATAATTATCAAAAGAGAGGGGTGGCAAACGGTATTTGGCATTATTAGGTTAAAAAATGTAGAAGGAGAGTGAAACCCATGAAAAAAATAATGCTAGTTTTTATTACACTTATATTAGTTAGTCTACCAATTGCGCAACAAACTGAAGCAAAGGATGCATCTGCATTCAATAAAGAAAATTCAATTTCAAAAAGGGCCGGCCCGAACGGCCACCTCATGGACGTCCACGGCAGCGGCAGCTACCCGGCACTGTAGTTCCAGGTTAAGGTTTCACCACCACGGTTGAGCGGGCGGGACATGGACGAGCTGCCGGACGCGCGCGTCCTGCTGAAGTCGCCGACCCTGTAGTTGTGGGGCCGCAAGCTCACCATGCTGGTCAGCCCGGACAGCCACCAGTACGGCCACCCACCGGTCAGTTCGAAGATGAGGCTGACCATGGTCGGGCGGACGCCGTTCCGGCCAACGGTCTGAATGTTCACCCTCTGGAAGGACTGGTCGCTCGACGGCCCCACCGACGTCCGGTTGTCCGTGCAGTTCGGGTGGCCTTCGCGGCAGCAGCCAGAAAGCTACCGACGAAGAAGCCGCGACTGCGACCGCTAGATAGTGGGGGTCGTCAAGCAGATGCGCCCTCGCTACAGCCCGGACAACCTGGGGAGGGTCCGCT ACCCAGGGTGGGACTAGCCGGACCGCTACCCACTGCGACCGCCGATGTTCCGGAGGCTGTACACCCCGGGCTTCCTCCTGGGCCGCACCGTCGCGTTGCTGGGCGACAACTTGCAGCCCTTCGACTAGCGGTTGTTGTGGGCGCAGACCCACATGACGCCGTTGCCGTTCGGCAGCCTAGACCCACCGTTGTTGGACGGCCGGTTCAAGGAGCTCCCGAAGCACGCCTGGTCGTTGTAGTTCAAGGTTCTGCGGATGTTGCGGCCACCGCCGGTGTTGCCGCACAAGCTGAAGGGCCTGTCGCCATGCGTGTCGACCCTCATGACCCCCCGCGTCGAGTTGCGATACTTCGGGCTGGACGTTGCCCGTGACCCACGGTGCGGGTTGTGGCCCGGGCGCGGGGTCCCGCGGCCGCCACCGCCTAGGCCACCGCCACCGAGGTGGTGGCGTGCGCTGTAGTACTTGCGTCCACACTGGACACAACCGCTTGTGCTCTGCGATTGGCGACGGCGAGTTATGTACGCACTCGTGCTGTAGCCGCGCAACGGCTAGACGCCCCTGCTGCTGGCCGACGTGCCGTACGAGTGGCTGGCGCTGTAACACTAGTTTCCGGACCGACGCCCGGATCTGGGCTTATGGCGGTGCCGACCGCTCAACCGGGCCCTGTCGTAGATGATGCAGCTACGCTTGCGTTCGTAGGTCCTCTACGAGTTGCAGTACCTTCTTGTAGTCCAGGCGGCACAAGGCCAGTAGAGTCTCGTGGCGAACCAGCCTTAGCAGTGGCTTCGGCTGTAGCGGGCTGTGGACGGGCTCGTGCGGTAACACGTCAAGCAGTTCCGTTAGACGAGCGGGTACCGGGAGCGGTCGCCAATCGAAAAGAAACACGCGGATGAAATCGATAAGTATATACAAGGATTGGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGAAAAGGTTACAAAGATGGAAA TGAATATATTGTTGTGGAGAAAAAGAAGAAATCCATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCAATTTCGAGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAATCAACCAGATGTTCTCCCTGTAAAACGTGATTCATTAACACTCAGCATTGATTTGCCAGGTATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCCACTAAATCAAACGTTAACAACGCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCAAATGTAAGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAATCACAATTAATTGCGAAATTTGGTACAGCATTTAAAGCTGTAAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGT。

The seventh aspect of the present invention provides the use of the fusion protein or its coding gene or the recombinant attenuated Listeria monocytogenes in the preparation of tuberculosis vaccine.

Further, the tuberculosis vaccine is a vaccine against dormant Mycobacterium tuberculosis.

The eighth aspect of the present invention provides a vaccine comprising the fusion protein or the recombinant attenuated Listeria monocytogenes.

The vaccine may further include an adjuvant.

The invention utilizes the property that the Listeria monocytogenes can survive in the phagocytosis, and can "escape" from the phagosome, enter the cytoplasm to reproduce, and induce the body to generate a strong CD8 ⁺ T cell immune response. Using homologous recombination technology, the foreign fusion protein gene is inserted into the genome of the vector organism. The vaccine provided by fusion expressing the early secretory protein Ag85a and the latent antigen Rv2626c regulated by DosR can effectively improve the immune protection effect of the host against mycobacteria, and provides a new idea for a new type of tuberculosis vaccine.

Description of drawings

Figure 1—Xba I and BamH I double-enzyme electrophoresis of pKSV7-hlya-Ag85a;

1:DL2000Marker

2: pKSV7-hlya-Rv2626c double enzyme digestion results

Figure 2—Xba I and Sal I double-enzyme electrophoresis of pKSV7-hlya-Ag85a-Rv2626c-hlyb;

M: λ-14 Marker

1: pKSV7-Ag85a-Rv2626c-hly double enzyme digestion results

Figure 3 - the identification results of the recombinant bacteria;

1: λ-14 Marker

2: LM4PCR product

3,4:rLM4PCR product

5:DL2000Marker

Figure 4 - Detection of the expression of the target gene at the protein level

M: Pre-stained marker

1: Secreted protein of rLm4

2: Secreted protein of LM4

Figure 5 - Verification of LLO expression

1: DL2000 Marker

2: yzuLM4RTPCR results

3: rLM4RTPCR results

4: λ-14 Marker

Figure 6 - Hemolytic activity test of secreted fusion protein LLO-Ag85a-Rv2626c

PBS: PBS control group

LM4: Listeria monocytogenes yzuLM4 group

rLm4: recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c group

Figure 7 - the results of recombinant bacteria on the cytokines induced by C57BL/6 mice.

Control: negative control

BCG: BCG

Ck: blank control

LM4: Listeria monocytogenes yzuLM4 group

rLm4: recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c group

Detailed ways

The embodiment of the present invention adopts following technical scheme:

First, Ag85a (also known as Ag85A, FbpA, Rv3804c) and Rv2626c fragments were amplified from the genome of Mycobacterium tuberculosis standard strain H37Rv, and hly upstream and downstream fragments were amplified from Listeria monocytogenes strain yzuLM4, The hlya-Ag85a-Rv2626c-hlyb fusion fragment obtained after gel recovery was ligated with the pMD-20T vector, transformed into Escherichia coli DH5α competent cells, and the correct positive clones verified by PCR and double enzyme digestion were sent to Nanjing KingScript And sequenced; extract the plasmid from the positive clone with correct sequencing, and then double enzyme digestion, recover the target fragment, then connect with the shuttle vector pKSV7, transform into E. coli DH5α competent cells, and verify the correctness by PCR and double enzyme digestion, the positive The plasmid was electrotransformed into yzuLM4 competent cells, screened by resistance and temperature double pressure, and then passaged without resistance to obtain homologous recombination Listeria monocytogenes without resistance gene. The bacteria is a recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c. The bacteria can express Ag85a-Rv2626c-LLO protein under normal living conditions.

The specific method for constructing recombinant attenuated bacteria mainly includes the following steps:

1. The target genes Ag85a, Rv2626c and the upstream and downstream homologous fragments hlya and hlyb of the site to be inserted into the hly gene were amplified by PCR technology.

The primers used are as follows:

Sense: 5'-ATAAAGAAAATTCAATTTCATTTTCCCGGCCGGGCTTG-3' (SEQ ID NO: 12)

Antisense: 5'-AATGCTGGATCCGCCACCGCCGGCGCCCTGGGGCGCGGGCA-3' (SEQ ID NO: 13)

The Ag85a gene with a length of 888 bp was recovered.

Sense: 5'-ATTGGATCCGGTGGCGGTGGCTCCACCACCGCACGCACAT-3' (SEQ ID NO: 14)

Antisense: 5'-CGTGTTTCTTTTCGATTGGCTGGCGAGGGCATG-3' (SEQ ID NO: 15)

The Rv2626c gene with a length of 420 bp was recovered.

Sense: 5'-AACCT GAGCTC AGGTTTGTTGTGTCAGGTAGAGC-3 (SEQ ID NO: 16)

Antisense: 5'-CAAGCCCGGCCGGGAAAATGAAATTGAATTTTCTTTAT-3' (SEQ ID NO: 17)

A 525bp hlya fragment was recovered.

Sense: 5'-GCCCTCGCCAGCCAATCGAAAAGAAACACG-3' (SEQ ID NO: 18)

Antisense: 5'-AATCA GTCGAC ACTTGAGATATATGCAGGAGG-3' (SEQ ID NO: 19)

A 732bp hlyb fragment was recovered.

2. Then use SOEing PCR technology to splice the four together, and then use the restriction site to insert the spliced fragment into the shuttle vector pKSV7.

The method of hlya fragment and Ag85a gene SOEing PCR splicing into hlya-Ag85a: Use sense and antisense primers to perform SOEing PCR splicing on the purified hlya fragment and Ag85a gene.

Sense: 5'-AACCT GAGCTC AGGTTTGTTGTGTCAGGTAGAGC-3 (SEQ ID NO: 16)

Antisense: 5'-AATGCT GGATCC GCCACCGCCGGCGCCCTGGGGCGCGGGCA-3' (SEQ ID NO: 13)

A hlya-Ag85a fragment with a length of 1208bp was recovered.

Rv2626c fragment and hlyb fragment SOEing PCR splicing into Rv2626c-hlyb method: Use sense and antisense primers to perform SOEing PCR splicing on the purified Rv2626c fragment and hlyb.

Sense: 5'-ATTGGATCCGGTGGCGGTGGCTCCACCACCGCACGCACAT-3' (SEQ ID NO: 14)

Antisense: 5'-GGGTCTAGAACTTGAGATATATGCAGGAGG-3' (SEQ ID NO: 19)

The 1125bp Rv2626c-hlyb fragment was recovered.

The method of inserting the hlya-Ag85a fragment into the shuttle vector pKSV7: double-digest the recovered hlya-Ag85a fragment with Xba I and BamH I enzymes, and simultaneously use Xba I and BamH I to double-digest the shuttle vector pKSV7, and then insert the hlya-Ag85a fragment with The carrier is connected. The method of inserting the Rv2626c-hlyb fragment into the shuttle vector pKSV7-hlya-Ag85a: double-digest the recovered Rv2626c-hlyb fragment with BamH I and Sal I enzymes, and simultaneously use BamH I and Sal I to double-digest the shuttle vector pKSV7-hlya- Ag85a, and then connect the Rv2626c-hlyb fragment to the carrier.

3. The recombinant plasmid pKSV7-hlya-Ag85a-Rv2626c-hlyb was introduced into Listeria monocytogenes by electrotransformation, homologous recombination was achieved under the double selection pressure of antibiotics and temperature, and the non-resistant gene was obtained by non-resistant passage Recombinant attenuated Listeria monocytogenes.

Secondly, Western-blotting experiment was performed on the secreted protein of recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c obtained. The primary antibody was mouse immunoprotein Ag85a and Rv2626c positive serum, the secondary antibody was goat anti-rabbit IgG labeled with horseradish peroxidase, and the chromogenic reagents were DAB and ECL. The results showed that the protein Ag85a-Rv2626c secreted by LMΔhly::Ag85a-Rv2626c and Ag85a and Rv2626c positive sera had specific immunological reactions.

At the same time, the safety of the obtained recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c was preliminarily evaluated, and the results showed that compared with the yzuLM4 strain, the obtained recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c had significantly lower toxicity, indicating that it was safe Sexually it is reliable.

Further, the immune protection of the obtained recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c was evaluated, and the results showed that the obtained recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c can induce mice to Mycobacterium tuberculosis protein Ag85a- Rv2626c produced a strong immune protective effect, and was more inclined to Th1-type immune response.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, devices and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology. These techniques have been fully described in the existing literature. For details, see Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York，1987and periodic updates；the series METHODS INENZYMOLOGY，Academic Press，San Diego；Wolffe，CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

The construction of embodiment 1 genetically engineered bacteria

1.1 Design of primers

According to the Ag85a and Rv2626c sequences of the genomic DNA of the standard Mycobacterium tuberculosis strain H37Rv, a pair of specific primers were designed respectively. The primers were synthesized by Beijing Liuhe Huada Gene, and the primers were as follows:

Ag85aF: 5'-ATAAAGAAAATTCAATTTCATTTTCCCGGCCGGGCTTG-3' (SEQ ID NO: 12)

Ag85aR: 5'- AATGCTGGATCCGCCACCGCCGGCGCCCTGGGGCGCGGGCA -3' (SEQ ID NO: 13)

Note: The underline represents the BamH I restriction site

Rv2626cF: 5'-ATT GGATCC GGTGGCGGTGGCTCCACCACCGCACGCACAT-3' (SEQ ID NO: 14)

Note: The underline represents the BamH I restriction site

Rv2626cR: 5'-CGTGTTTCTTTTCGATTGGCTGGCGAGGGCATG-3' (SEQ ID NO: 15)

According to the LLO upstream and downstream sequences of the genomic DNA of Listeria monocytogenes yzuLM4, a pair of specific primers were respectively designed. The primers were synthesized by Beijing Liuhe Huada Gene. The primers were as follows:

hlyaF:5'-AACCT GAGCTC AGGTTTGTTGTGTCAGGTAGAGC-3' (SEQ ID NO: 16)

Note: The underline represents the Xba I restriction site

hlyaR:5'-CAAGCCCGGCCGGGAAAATGAAATTGAATTTTCTTTAT-3' (SEQ ID NO: 17)

hlybF:5'-GCCCTCGCCAGCCAATCGAAAAGAAACACG-3' (SEQ ID NO: 18)

hlybR: 5'-AATCA GTCGAC ACTTGAGATATATGCAGGAGG-3' (SEQ ID NO: 19)

Note: The underline represents the Sal I restriction site

1.2 PCR amplification of target gene, product recovery and construction of cloning vector

Genomic DNA of Mycobacterium tuberculosis standard strain H37Rv and Genomic DNA of Listeria monocytogenes strain yzuLM4 were extracted using the Genomic DNA Extraction Kit of Tiangen Bacteria, respectively, using them as templates, Ag85aF, Ag85aR; Rv2626cF, Rv2626cR; hlyaF, hlyaR; hlybF and hlybR were used as primers for PCR amplification. The PCR reaction system is 50 μL:

Sterilized ultrapure water: 37.5μL

10×PCR Buffer: 5 μL

Upstream primer (100 μmol/L): 1 μL

Downstream primer (100 μmol/L): 1 μL

Taq enzyme (3U/μL): 0.5 μL

dNTP (2.5mmol/L): 4μL

Bacterial genome template: 1 μL

The PCR reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 40 s, annealing at 58°C for 40 s, extension at 72°C for 1 min, 30 cycles of 40 s, 40 s, and 50 s, and extension at 72°C for 10 min. The PCR product was separated by agarose gel electrophoresis at a concentration of 1%, and the DNA fragments of 888bp, 420bp, 525bp, and 732bp were recovered with the Biotech Multifunctional DNA Purification and Recovery Kit, and the fragments recovered by Ag85a, hlya, Rv2626c, and hlyb were respectively Template, hlyaF, Ag85aR and Rv2626cF, hlybR are used as primers for PCR amplification, and the PCR reaction system is 50 μL:

Sterilized ultrapure water: 34.5 μL

10×PCR Buffer: 5 μL

Upstream primer (100 μmol/L): 1 μL

Downstream primer (100 μmol/L): 1 μL

Taq enzyme (3U/μL): 0.5 μL

dNTP (2.5mmol/L): 4μL

Recovered fragment template: 4 μL

The PCR reaction conditions were: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 40 seconds, annealing at 55°C for 1 minute, extension at 72°C for 80 seconds, 30 cycles, and extension at 72°C for 10 minutes. The PCR product was separated by agarose gel electrophoresis at a concentration of 1%, and the DNA fragments of 1208bp and 1125bp were recovered with the Biotec Multifunctional DNA Purification and Recovery Kit, and then respectively connected to the pMD-20T carrier in a metal bath at 16°C overnight, transformed DH5α competent cells, positive clones were screened by PCR and double enzyme digestion verification. The obtained positive clones were sent to Nanjing GenScript for sequencing, and the correctly sequenced plasmids were named pMD-20T-hlya-Ag85a and

pMD-20T-Rv2626c-hlyb.

Among them, the sequencing result of pMD-20T-hlya-Ag85a is SEQ ID NO: 20:

ACGCCAAGCTATTTAGGTGACACTATAGGGGAAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCTACTAGTCATATGGATAATGCTGGATCCGCCACCGCCGGCGCCCTGGGGCGCGGGCCCGGTGTTGGGCGTGGCACCCAGTGCCCGTTGCAGGTCGGGCTTCATAGCGTTGAGCTGCGCCCCCCAGTACTCCCAGCTGTGCGTACCGCTGTCCGGGAAGTCGAACACGCCGTTGTGGCCGCCACCGGCGTTGTAGGCGTCTTGGAACTTGATGTTGCTGGTCCGCACGAAGCCCTCGAGGAACTTGGCCGGCAGGTTGTTGCCACCCAGATCCGACGGCTTGCCGTTGCCGCAGTACACCCAGACGCGGGTGTTGTTGGCGATCAGCTTCCCGACGTTCAACAGCGGGTCGTTGCGCTGCCACGCCGGGTCCTCCTTCGGGCCCCACATGTCGGAGGCCTTGTAGCCGCCAGCGTCACCCATCGCCAGGCCGATCAGGGTGGGACCCATCGCCTGGGAGGGGTCCAACAGGCCCGACATCGCTCCCGCGTAGACGAACTGCTGGGGGTGATAGATCGCCAGCGTCAGCGCCGAAGAAGCAGCCATCGAAAGACCGACGACGGCGCTTCCGGTGGGCTTGACGTGCCTGTTGGCCTGCAGCCACCCCGGCAGCTCGCTGGTCAGGAAGGTCTCCCACTTGTAAGTCTGGCAACCGGCCTTGCCGCAGGCGGGCTGGTACCAGTCGGAGTAGAAGCTTGACTGGCCACCCACCGGCATGACCACCGACAGGCCCGACTGGTCGTACCACTCGAACGCCGGGGTGTTGATGTCCCAGCCGCTGAAGTCGTCCTGCGCGCGCAGGCCGTCGAGCAGGTACAGGGCGGGCGAGTTGGCACCACCACTTTGGAATTGGACCTTGATGTCACGGCCCATCGACGGCGACGGCACCTGCAGGTACTCCACCGGCAAGCCCGGCCGGGAAAATGAAATTGAAT TTTCTTTATTGAATGCAGATGCATCCTTTGCTTCAGTTTGTTGCGCAATTGGTAGACTAACTAATATAAGTGTAATAAAAACTAGCATTATTTTTTTCATGGGTTTCACTCTCCTTCTACATTTTTTAACCTAATAATGCCAAATACCGTTTGCCACCCCTCTCTTTTGATAATTATAATATTGGCGAAATTCGCTTCTAAAGATGAAACGCAATATTATATGCTTGCTTTATAGCTTTATTCTAGTCCTGCTGTCCCTTTATCGTCGTTAACAAATGTTAATGCCTCAACATAAAAGTCACTTTAAGATAGGAATATACTAATCAAAGGAGGGGGCCATTTTGTATAAGAATTATTTGCAACGCACATTAGTTTTATTACTCTGTTTTATTTTATACTTTTTTACTTTCCCATTAGGCGGAAAAGCATATTCGCTTAATAACTGGAATAAGCCAATAAAGAACTCTGTAACTACAAAACAATGGATGTCCGCTCTACCTGACACAACAAACCTGAGCTCAGGTATCGGATCCCCGGGTACCGAGCTCGAATTC

The sequencing result of pMD-20T-Rv2626c-hlyb is SEQ ID NO:21::

AGCTCGGTACCCGGGGATCCGATTGGATCCGGTGGCGGTGGCTCCACCACCGCACGCGACATCATGAACGCAGGTGTGACCTGTGTTGGCGAACACGAGACGCTAACCGCTGCCGCTCAATACATGCGTGAGCACGACATCGGCGCGTTGCCGATCTGCGGGGACGACGACCGGCTGCACGGCATGCTCACCGACCGCGACATTGTGATCAAAGGCCTGGCTGCGGGCCTAGACCCGAATACCGCCACGGCTGGCGAGTTGGCCCGGGACAGCATCTACTACGTCGATGCGAACGCAAGCATCCAGGAGATGCTCAACGTCATGGAAGAACATCAGGTCCGCCGTGTTCCGGTCATCTCAGAGCACCGCTTGGTCGGAATCGTCACCGAAGCCGACATCGCCCGACACCTGCCCGAGCACGCCATTGTGCAGTTCGTCAAGGCAATCTGCTCGCCCATGGCCCTCGCCAGCCAATCGAAAAGAAACACGCGGATGAAATCGATAAGTATATACAAGGATTGGATTACAATAAAAACAATGTATTAGTATACCACGGAGATGCAGTGACAAATGTGCCGCCAAGAAAAGGTTACAAAGATGGAAATGAATATATTGTTGTGGAGAAAAAGAAGAAATCCATCAATCAAAATAATGCAGACATTCAAGTTGTGAATGCAATTTCGAGCCTAACCTATCCAGGTGCTCTCGTAAAAGCGAATTCGGAATTAGTAGAAAATCAACCAGATGTTCTCCCTGTAAAACGTGATTCATTAACACTCAGCATTGATTTGCCAGGTATGACTAATCAAGACAATAAAATCGTTGTAAAAAATGCCACTAAATCAAACGTTAACAACGCAGTAAATACATTAGTGGAAAGATGGAATGAAAAATATGCTCAAGCTTATCCAAATGTAAGTGCAAAAATTGATTATGATGACGAAATGGCTTACAGTGAATCACAATTAATTGCGAAATTTGGTACAGCATTTAAAGCTGT AAATAATAGCTTGAATGTAAACTTCGGCGCAATCAGTGAAGGGAAAATGCAAGAAGAAGTCATTAGTTTTAAACAAATTTACTATAACGTGAATGTTAATGAACCTACAAGACCTTCCAGATTTTTCGGCAAAGCTGTTACTAAAGAGCAGTTGCAAGCGCTTGGAGTGAATGCAGAAAATCCTCCTGCATATATCTCAAGTTCTAGATGATATCCATATGACTAGTAGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTTCCCCTATAGTGTCACCTAAATAGCTTGGCGTAAT

All in line with expectations.

1.3 Construction and identification of the shuttle vector pKSV7-hlya-Ag85a-Rv2626c-hlyb

After pMD-20T-hlya-Ag85a was digested with Xba I and BamH I, the digested product was separated by agarose gel electrophoresis at a concentration of 1%, and the DNA fragment of 1208bp was recovered with Biotech Multifunctional DNA Purification and Recovery Kit , and pKSV7 vector were connected overnight at 16°C in a metal bath, transformed into Escherichia coli DH5α competent cells, and positive clones were screened by PCR and double enzyme digestion verification, the results are shown in Figure 1. The verified correct plasmid was named pKSV7-hlya-Ag85a. After pMD-20T-Rv2626c-hlyb was digested by BamH I and Sal I, the digested product was separated by agarose gel electrophoresis at a concentration of 1%, and the DNA fragment of 1125bp was recovered with Biotech Multifunctional DNA Purification and Recovery Kit , connected with the pKSV7-hlya-Ag85a carrier overnight at 16°C in a metal bath, transformed E. coli DH5α competent cells, and screened the positive clone pKSV7-hlya-Ag85a-Rv2626c-hlyb by PCR and double enzyme digestion verification, the results are shown in the figure 2.

1.4 Construction of recombinant bacteria LM⊿hly::Ag85a-Rv2626c

The recombinant shuttle plasmid pKSV7-hlya-Ag85a-Rv2626c-hlyb was transformed into competent yzuLM4 by electroporation (2200v, 5ms). Inoculate in BHI medium, pass 8 consecutive generations under the double selection pressure of temperature (42°C) and chloramphenicol (10 μg/ml) to achieve homologous recombination, so that the target gene can be integrated into the bacterial genomic DNA. Passage for another 10 generations under the condition of no selection pressure to get rid of the shuttle plasmid. Dilute the bacterial solution and spread it on a BHI plate to grow a single colony at 30°C, then transfer the single colony to a BHI plate containing chloramphenicol (10 μg/ml) and culture at 30°C. Pick a single colony that grows on the BHI plate and no longer grows after transplantation, extracts the genome and conducts PCR identification. The positive clone is the recombinant LM4Δhly::Ag85a-Rv2626c.

The primers for the PCR reaction are:

hlyaF: ditto

hlyb+: 5-GTTTCATCATCACCTGAGACAGATTTTCCGC-3 SEQ ID NO: 22:

The PCR reaction system (50μl) is:

Sterilized ultrapure water: 37.5μL

10×PCR Buffer: 5 μL

Upstream primer (100 μmol/L): 1 μL

Downstream primer (100 μmol/L): 1 μL

Taq enzyme (3U/μL): 0.5 μL

dNTP (2.5mmol/L): 4μL

Bacterial genome template: 1 μL

The PCR reaction conditions were: 94°C for 5 minutes; 25 cycles of 94°C for 50s, 62°C for 30s, and 72°C for 2 minutes; 72°C for 10 minutes. The size of the PCR product detected by 1% agarose gel electrophoresis should be 2680bp, as shown in Figure 3.

It can be seen from the results in Figure 3 that the target gene fragment was successfully integrated into the genome of Listeria monocytogenes yzuLM4, and the plasmid was successfully removed under the non-resistant stress passage, and a non-resistant recombinant attenuated bacterium LMΔhly was obtained ::Ag85a-Rv2626c.

Example 2 Detection of the expression of the target gene at the protein level

Inoculate the LMΔhly::Ag85a-Rv2626c constructed in Example 1 into the BHI liquid medium, culture on a shaker at 37°C for 15 hours, take 1ml of the bacterial liquid, centrifuge to remove the bacterial cells, and collect the culture supernatant. The secreted protein obtained by the TCA precipitation method was subjected to SDS-PAGE electrophoresis. After the electrophoresis, the gel was taken out, the length and width were measured, and the NC membrane was cut out 0.5 cm shorter than the length and width of the gel. The length and width of the gel are 1cm shorter filter paper, soak them in the transfer buffer, and transfer them to the electrotransfer device according to the structure of filter paper-filter paper-gel-NC membrane-filter paper-filter paper, after the transfer is completed, take out the NC The membrane was placed in 1% BSA-PBS blocking solution and blocked overnight. Wash 3 times with PBS (PBST) containing 0.05% Tween20 at room temperature, dilute mouse immunoprotein Ag85a and Rv2626c positive sera at 1:1000 with blocking solution respectively as primary antibodies, shake at room temperature for 3 hours; wash 5 times with PBST, each time 5min; dilute horseradish peroxidase-labeled goat anti-mouse IgG with blocking solution at 1:3000 as the secondary antibody, shake at room temperature for 1h; wash 5 times with PBST, 5min each time; finally immerse the NC membrane in DAB ( 3,3-diaminobenzidine) solution and ECL (chemiluminescence substrate) color development, rinse with distilled water to terminate the reaction, the results are shown in Figure 4.

It can be seen from the results in Figure 4 that the target bands of about 70 kDa were obtained when the positive sera of mouse immunoprotein Ag85a and Rv2626c were used as primary antibodies, indicating that the target fragments Ag85a and Rv2626c were both correctly expressed.

In order to prove that LLO was correctly expressed, the total RNA of LMΔhly::Ag85a-Rv2626c and yzuLM4 bacteria were extracted respectively, and reverse transcription PCR was performed to design a pair of sequences spanning 1372bp starting from the start codon of the hly gene, including hly Most of the sequence of the gene, and the downstream primers are on the periphery of the hlybR primers used for homologous recombination:

F: ATGAAAAAAATAATGCTAGT (SEQ ID NO: 23):

R: GACGATGTGAAATGAGCTAGC (SEQ ID NO: 24).

The PCR reaction system (20μl) is:

Sterilized ultrapure water: 11.8μL

10×PCR Buffer: 2μL

Upstream primer (100 μmol/L): 1 μL

Downstream primer (100 μmol/L): 1 μL

Taq enzyme (3U/μL): 0.2 μL

dNTP (2.5mmol/L): 2μL

Bacterial RNA reverse transcription template: 2 μL

The results are shown in Figure 5.

As can be seen from the results in Figure 5, LMΔhly::Ag85a-Rv2626c has a band around 2600bp, which includes a partial fragment of the hly gene of 1359bp and the sequence of the fusion gene Ag85a-Rv2626c of 1308bp, while yzuLM4 only has a band of 1359bp in size bring. From the results in Figure 4 and Figure 5, it can be known that LLO was correctly expressed and the fusion gene was integrated at a specific point.

Example 3 Safety Evaluation of Recombinant Bacteria

3.1 Hemolytic activity test of secreted fusion protein LLO-Ag85a-Rv2626c

Inoculate the LMΔhly::Ag85a-Rv2626c and yzLM4 constructed in Example 1 to a quantitative volume of BHI liquid medium, culture on a shaker at 37°C for 15 hours, take 1ml of the bacterial liquid, centrifuge to remove the bacterial cells, and collect the culture supernatant. After adjusting the OD600 of the supernatant to the same size, take 60 μl of the supernatant and dilute it with PBS (pH6.0) from the stock solution to 27 equal times, add it to the V-shaped 96-well hemagglutination plate, and then add it to each well 20 μL of 1% goat erythrocyte suspension, mixed evenly, placed in a 37°C incubator for 1 hour to observe the hemolysis, and the dilution of the well where 50% of the erythrocytes were hemolyzed was judged as the hemolysis titer. PBS was used as the control group. The results are shown in Figure 6.

The results showed that the hemolytic activity of the Listeria monocytogenes yzuLM4 group was about 25, which had a strong hemolytic activity, while the recombinant attenuated strain LMΔhly::Ag85a-Rv2626c had no hemolytic activity. make it secure in terms of security. It provides a safe basis for the subsequent development of the recombinant attenuated bacteria.

3.2 Determination of LD50 of recombinant bacteria on C57BL/6 mice

Take the freshly cultivated recombinant bacteria liquid, wash it twice with PBS, measure the OD600 and adjust the OD600 to 0.80. The recombinant bacteria were serially diluted 10 times, and an appropriate dilution was used to infect 6-week-old male C57BL/6 mice. The infection method is as follows: 5 experimental mice are injected intravenously for each dilution, 100 μL/mouse, and 5 serial dilutions are made. At the same time, the bacteria were appropriately diluted and spread on the BHI culture plate for colony counting. After the infection was observed continuously for 14 days, the number of dead animals in each group was recorded, and the LD50 was calculated by the Reed-Muench method. The results are shown in the table below.

Table 1 Determination of LD50 of recombinant bacteria on C57BL/6 mice

It can be seen from the results in the above table that the LD50 value of the recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c is 1.35×10 ⁹ CFU/ml, and the toxicity to mice is significantly reduced, which is the safety of the recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c Guarantees are provided.

Evaluation of the immune protection effect of embodiment 4 recombinant bacteria

4.1 Immunization of mice

Prepare bacteria according to the method in Example 3.2, immunize 6-8 week old female C57BL/6 mice by tail vein, 3×10 ⁸ CFU/mouse, 6 mice in each group, and set PBS phosphate buffer solution (100 μL/mouse) for immunization at the same time Negative control group, wild Listeria monocytogenes yzuLM4 group, recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c (rLM4) group. The second immunization was carried out two weeks after the first immunization, and the mice were culled 8 days after the second immunization, and relevant immunological experiments were carried out.

4.2 Preparation of mouse spleen lymphocytes

The mice were killed by taking out the eyeballs and blood collection, and the spleens of the mice were aseptically removed from the biological safety cabinet, placed in a plate containing 3-5mL ice-bath medium (CM), and fully squeezed with the blunt end of a frosted glass slide. Press and grind the splenocytes to disperse into a single cell suspension; filter through a 200-mesh copper mesh, centrifuge at 1000rpm for 10min at 4°C, discard the supernatant, and resuspend the pellet with 2-3mL red blood cell lysate to lyse the red blood cells, 37 ℃ for 3-5 minutes, add 2 times the volume of PBS to stop; 4 ℃, 1000rpm centrifuge for 10 minutes, remove the supernatant, resuspend with fresh CM and centrifuge to wash the cells twice, finally resuspend the cells in CM, trypan blue Count after staining, adjust the cell concentration to 1×10 ⁷ cells/mL, and keep in ice bath for later use.

4.3 Sandwich ELISA quantitative detection of specific IFN-γ and IL-4 cytokines

4.3.1 Cell culture supernatant preparation

Adjust the spleen cell suspension of each group of mice prepared above to 1× ¹⁰⁶ cells/50 μL and add it to a 96-well cell plate, and then add 50 μL of Bovine PPD (bovine tuberculin) diluted with CM (10 μg/mL) Stimulate with the purified proteins Ag85a and Rv2626c (10 μg/mL) expressed in the corresponding prokaryotes, and set up a negative control (unstimulated cells). In order to prove the operational accuracy of this experiment, a blank control was set up at the same time. Multiple wells were incubated at 37°C in a 5% CO ₂ incubator for 48 hours. Centrifuge at 1000 rpm for 5 min at 4°C, and collect the cell culture supernatant for use.

4.3.2 Sandwich ELISA test

The day before the test, the ELISA plate was taken and coated with anti-mouse IFN-γmAb and IL-4mAb respectively, 2 μg/mL, 100 μL/well, overnight at 4°C; the next day, PBST (phosphate Tween buffer) was washed 5 times, and adding Block with 1% BSA in PBS at 37°C for 2 hours; wash with PBST 5 times, add the supernatant of each cell to be tested, 100 μL/well, and add commercial standard recombinant mouse IFN-γ and IL-4 from 2000 and 4000 pg/mL respectively Start to make serial doubling dilutions as a standard curve control, let stand at room temperature for 3 hours; wash 5 times with PBST, add corresponding biotinylated detection antibodies IFN-γ-biotin mAb, IL-4-biotin mAb, 1 μg/mL, 100uL/well, place at room temperature for 1h; wash 5 times with PBST, dilute streptavidin-labeled horseradish peroxidase (streptavidin HRP) with PBS containing 1%BSA 1:1000, 100μL/well, place at room temperature for 30min; wash with PBST 7 times, add TMB substrate for color development, stop with 2M H ₂ SO4, read the OD ₄₅₀ value with an enzyme-linked immunosorbent reader, and calculate the contents of IFN-γ and IL-4 cytokines in the supernatant of cells in each group according to the drawn standard curve (pg/mL).

The result is shown in Figure 7:

After two immunizations of PBS negative control group, wild strain yzuLM4 without inserted tuberculosis antigens Ag85a and Rv2626c, and recombinant strain LMΔhly::Ag85a-Rv2626c expressing Ag85a and Rv2626c mice, the spleen cells of the mice in the corresponding immunized groups were taken out and placed in In vitro, the purified proteins Ag85a and Rv2626c expressed in prokaryotic and tuberculin (a mixture containing wild-type Mycobacterium tuberculosis secretory protein, PPD) were used to stimulate and incubate the IFN-r and IL- 4 The results of content determination showed that: 1) The contents of these two cytokines secreted by the PBS negative control group and the wild strain yzuLM4 without tuberculosis antigens Ag85a and Rv2626c were extremely low, while the recombinant strain LMΔhly::Ag85a-Rv2626c expressing Ag85a and Rv2626c secreted The levels of the above two cytokines were extremely high and showed significant differences, which indicated that LMΔhly::Ag85a-Rv2626c immunized mice to induce specific immune responses against tuberculosis fusion proteins Ag85a and Rv2626c. 2) The IFN-r secretion level (1000pg/mL) of the recombinant attenuated bacteria LMΔhly::Ag85a-Rv2626c group was significantly higher than that of IL-4 (250pg/mL), which indicated that the type of immune response induced by the recombinant bacteria was more inclined to Th1-type immune response, that is, cellular immune response. 3) The recombinant vaccine LMΔhly::Ag85a-Rv2626c can not only induce the cellular immunity of mice against the early secretory protein Ag85a of Mycobacterium tuberculosis, but also effectively induce the cellular immune response of Rv2626c, which is expressed when Mycobacterium tuberculosis is latently infected in vivo . In summary, LMΔhly::Ag85a-Rv2626c immunized mice can induce a Th1-type cellular immune response against the fusion protein Ag85a-Rv2626c. prevention has potential application value.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method and composition of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (14)

1. A fusion protein having immunogenicity for tuberculosis, is a fusion protein containing mycobacterium Ag85a and Rv2626c; said fusion protein is monocytogenes listeria hemolysin protein LLO and mycobacterium Ag85a and Rv2626c fusion protein; in the fusion protein, the fusion protein of Ag85a protein and Rv2626c protein is inserted between the 37th and 38th amino acid residues of the monocytogenes listeria hemolysin protein LLO; the The amino acid sequence of the fusion protein is SEQ ID NO:4. 2. fusion protein as claimed in claim 1, is characterized in that, in described fusion protein, the amino acid sequence of Ag85a is SEQ ID NO:1; The amino acid sequence of Rv2626c is SEQ ID NO:2. 3. fusion protein as claimed in claim 1, is characterized in that, the aminoacid sequence of described monocytogenetic listeria hemolysin protein LLO is SEQ ID NO:3. 4. A polynucleotide encoding the fusion protein according to any one of claims 1-3. 5. A vector comprising the polynucleotide according to claim 4. 6. The vector according to claim 5, wherein the vector is a prokaryotic vector or a shuttle plasmid. 7. A host cell transformed with the vector according to claim 5 or 6. 8. A recombinant attenuated monocytogenes listeria, in the genome of said recombinant attenuated monocytogenes listeria, integrated with the gene of the fusion protein of encoding mycobacterium Ag85a and Rv2626c, said Recombinant attenuated monocytogenes Listeria can fuse and express the fusion protein of monocytogenes listeria hemolysin protein LLO and mycobacterium Ag85a and Rv2626c; said fusion protein encoding mycobacteria Ag85a and Rv2626c Gene recombination is integrated between bases 111 and 154 of the hly gene in the genomic DNA of wild-type Listeria monocytogenes; compared with wild-type Listeria monocytogenes, the recombinant attenuated monocytogenes In Listeria monocytogenes, the hly coding gene of wild-type Listeria monocytogenes was replaced by the coding gene of the fusion protein whose sequence is SEQ ID NO:4. 9. recombinant attenuated monocytogenes listeria as claimed in claim 8, is characterized in that, in the fusion protein of described mycobacterium Ag85a and Rv2626c, the aminoacid sequence of Ag85a is SEQ ID NO:1, that of Rv2626c The amino acid sequence is SEQ ID NO:2. 10. recombinant attenuated monocytogenes listeria as claimed in claim 8, is characterized in that, the nucleotide sequence of the gene of the fusion protein of described coding mycobacterium Ag85a and Rv2626c is SEQ ID NO:5. 11. The construction method of recombinant attenuated monocytogenes Listeria bacterium according to any one of claims 8-10, comprising the following steps: 1) Amplify the coding gene fragments of Ag85a and Rv2626c from the genome of Mycobacterium tuberculosis strain; amplify the upstream homology arm fragment hlya and the downstream homology arm of the insertion site from the Listeria monocytogenes strain fragment hlyb; 2) Splicing the coding genes of Ag85a and Rv2626c and the upstream and downstream homology arm fragments obtained in step 1) into a hlya-Ag85a-Rv2626c-hlyb fusion fragment and connecting them into a shuttle plasmid to obtain a recombinant shuttle plasmid; 3) Transform the recombinant shuttle plasmid obtained in step 2) into Listeria monocytogenes, screen for resistance and temperature double pressure, and then obtain recombinant attenuated monocytogenes without resistance gene by passage without resistance Listeria bacteria. 12. The construction method of recombinant attenuated monocytogenes listeria as claimed in claim 11, is characterized in that, in step 1), the sequence of the upstream homology arm fragment hlya that amplifies is SEQ ID NO:6 The sequence of the downstream homology arm fragment hlyb is SEQ ID NO: 7; the coding gene of Ag85a is SEQ ID NO: 8; the coding gene of Rv2626c is SEQ ID NO: 9. 13. The fusion protein or its encoding gene according to any one of claims 1-3 or the recombinant attenuated monocytogenes Listeria in the preparation of tuberculosis vaccine according to any one of claims 8-10 use. 14. A vaccine comprising the fusion protein according to any one of claims 1-3 or the recombinant attenuated Listeria monocytogenes according to any one of claims 8-10.