阅读说明：本技术 一种密码子优化的基因、重组表达载体、重组蛋白及多克隆抗体的制备方法和应用 (Codon-optimized gene, recombinant expression vector, recombinant protein and polyclonal antibody preparation method and application ) 是由 许涛 汪洪涛 钱中清 李敏英 王楚彤 袁美丽 于 2021-09-09 设计创作，主要内容包括：本发明涉及基因工程技术领域,特别是涉及一种密码子优化的基因、重组表达载体、重组蛋白及多克隆抗体的制备方法和应用。本发明提供了一种密码子优化的PE-PGRS45基因,所述PE-PGRS45基因核苷酸序列如SEQ ID NO.1所示。利用本发明所述PE-PGRS45基因能够用于制备效价高的多克隆抗体。本发明具体实施例中试验结果表明,该基因的密码子适应指数(CAI)从0.73增加到0.90。(The invention relates to the technical field of genetic engineering, in particular to a preparation method and application of a codon optimized gene, a recombinant expression vector, a recombinant protein and a polyclonal antibody. The invention provides a PE _ PGRS45 gene with optimized codons, wherein the nucleotide sequence of the PE _ PGRS45 gene is shown as SEQ ID NO. 1. The PE _ PGRS45 gene can be used for preparing high-titer polyclonal antibodies. The results of the experiments in the specific examples of the present invention show that the Codon Adaptation Index (CAI) of the gene increases from 0.73 to 0.90.)

1. A PE _ PGRS45 gene optimized by codons is characterized in that the nucleotide sequence of the PE _ PGRS45 gene is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising the gene of claim 1, wherein the base plasmid of the recombinant expression vector comprises pMAL-c5 x.

3. The recombinant expression vector according to claim 2, wherein the nucleotide sequence of said gene is located between Nde I and Hind iii cleavage sites of said pMAL-c5 x.

4. A recombinant protein is characterized in that the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 8.

5. A recombinant protein is characterized in that the sequence of the coding gene of the recombinant protein comprises the sequence shown in SEQ ID NO. 1.

6. A method for producing a recombinant protein according to claim 4 or 5, comprising the steps of:

the recombinant expression vector of claim 2 or 3 is used to transform Escherichia coli, and then the Escherichia coli is cultured and induced in sequence to obtain recombinant protein.

7. Root of herbaceous plantThe production method according to claim 6, wherein in the culturing, an LB liquid medium containing an antibiotic is used as the culture medium; the culture temperature is 20-37 ℃, and the culture finishing standard is OD of the bacterial liquid obtained by culture₆₀₀＝0.4～0.6；

At induction, the medium used included the following components at the following concentrations: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50ug/mL kanamycin and 0.5mM IPTG;

the induction temperature is 20-37 ℃, the rotating speed is 200r/min, and the time is 12 h.

8. A method for preparing a polyclonal antibody with high titer is characterized by comprising the following steps:

a host is immunized by using the recombinant protein of claim 4 or 5 as an antigen to obtain a polyclonal antibody against PE _ PGRS 45.

9. Use of the polyclonal antibody prepared by the preparation method of claim 8 in the preparation of a kit for diagnosing tuberculosis.

10. A kit for diagnosing tuberculosis, comprising the polyclonal antibody prepared by the preparation method of claim 7.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a preparation method and application of a codon optimized gene, a recombinant expression vector, a recombinant protein and a polyclonal antibody.

Background

Mycobacterium tuberculosis (Mycobacterium tuberculosis) is the main intracellular bacterium responsible for tuberculosis. Because of the low efficacy of bacillus calmette-guerin (BCG), the drug resistance caused by the abuse of antibiotics is enhanced, so that tuberculosis threatens the global health again. According to the statistics of the World Health Organization (WHO), 1000 ten thousand new cases are added in 2019 worldwide, 141 ten thousand patients die, and the death is the leading factor of a single infectious disease.

The mycobacterium tuberculosis PE _ PGRS45 protein is encoded by Rv2615c gene and has high homology with PE _ PGRS17 (Rv0978c) and PE _ PGRS18(Rv0980 c). Bioinformatics analysis shows that serine residues of a conserved tetrapeptide motif (DEVS/DXXS) of PE _ PGRS45 can be competitively combined with caspase-3 protein after being phosphorylated and participate in programmed death of host cells, and the PE _ PGRS45 is suggested to have important significance in mycobacterium tuberculosis infection. Therefore, it is urgent to seek a method for obtaining high titer polyclonal antibody from the PE _ PGRS45 gene.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method and application of a codon optimized gene, a recombinant expression vector, a recombinant protein and a polyclonal antibody. The PE _ PGRS45 gene is subjected to codon optimization, so that the PE _ PGRS45 recombinant protein can be obtained, and a high-titer and high-polyclonal antibody can be obtained.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a PE _ PGRS45 gene with optimized codons, wherein the nucleotide sequence of the PE _ PGRS45 gene is shown as SEQ ID NO. 1.

The invention provides a recombinant expression vector containing the gene, and the basic plasmid of the recombinant expression vector comprises pMAL-c5 x.

Preferably, the nucleotide sequence of said gene is located between Nde I and Hind III cleavage sites of said pMAL-c5 x.

The invention provides a recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 8.

The invention provides a recombinant protein, wherein the sequence of the coding gene of the recombinant protein comprises a sequence shown in SEQ ID NO. 1.

The invention provides a preparation method of the recombinant protein, which comprises the following steps:

after the recombinant expression vector is used for transforming escherichia coli, the recombinant expression vector is sequentially cultured and induced to obtain recombinant protein.

Preferably, the culture medium adopted during the culture is LB liquid culture medium containing antibiotics; the culture temperature is 20-37 ℃, and the culture finishing standard is OD of the bacterial liquid obtained by culture₆₀₀＝0.4～0.6；

At induction, the medium used included the following components at the following concentrations: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50ug/mL kanamycin and 0.5mM IPTG;

the induction temperature is 20-37 ℃, the rotating speed is 200r/min, and the time is 12 h.

The invention provides a preparation method of a polyclonal antibody with high titer, which comprises the following steps:

the recombinant protein is used as an antigen to immunize a host, so as to obtain the polyclonal antibody against PE _ PGRS 45.

The invention provides application of the polyclonal antibody prepared by the preparation method in preparing a kit for diagnosing tuberculosis.

The invention provides a kit for diagnosing tuberculosis, which comprises the polyclonal antibody prepared by the preparation method.

The invention provides a PE _ PGRS45 gene with optimized codons, wherein the nucleotide sequence of the PE _ PGRS45 gene is shown as SEQ ID NO. 1. The PE _ PGRS45 gene can be used for obtaining a polyclonal antibody with high titer. The results of the experiments in the specific examples of the present invention show that the Codon Adaptation Index (CAI) of the gene increases from 0.73 to 0.90.

Drawings

Fig. 1 is a comparison of the homologous amino acid sequences of mycobacterium tuberculosis PE _ PGRS45, wherein m.tubericalis is mycobacterium tuberculosis, m.bovis is mycobacterium bovis, m.africanum is mycobacterium africanum, m.canettii is mycobacterium tryantii, m.caprae is mycobacterium capricae, and the lowest sequences in each row are the same sequences as m.tubericalis, m.bovis, m.africanum, m.canettii and m.caprae 5;

FIG. 2 is a diagram showing codon analysis of Mycobacterium tuberculosis PE _ PGRS45 gene;

FIG. 3 shows the results of recombinant expression vector identification, wherein A is the result of restriction of recombinant expression vector pET-28a-PE _ PGRS4 by Nco I and Xho I, M is DNA marker, 1 is the result of restriction of recombinant expression vector pET-28a-PE _ PGRS4 by Nco I and Xho I, B is the result of restriction of recombinant expression vector pET-32a-PE _ PGRS45 by Nco I and Xho I, M is DNA marker, 1 is the result of restriction of recombinant expression vector pET-32a-PE _ PGRS45, C is the result of restriction of recombinant expression vector pMAL-C5x-PE _ PGRS45 by Nde I and Hind III, M is DNA marker, 1 is the result of recombinant expression vector pMAL-C5x-PE _ PGRS 45;

FIG. 4 is a SDS-PAGE analysis of recombinant expression vectors pET-28a-PE _ PGRS45 and pET-32a-PE _ PGRS45 after codon optimization, where A is the fusion expression of pET-28a-PE _ PGRS45 in Escherichia coli at 20 ℃, B is the fusion expression of pET-28a-PE _ PGRS45 in Escherichia coli at 37 ℃, C is the fusion expression of pET-32a-PE _ PGRS45 in Escherichia coli at 20 ℃, D is the fusion expression of pET-32a-PE _ PGRS45 in Escherichia coli at 37 ℃, M is a protein marker, 1 is an E.coli cell before induction, 2 is an E.coli cell after induction, 3 is a lysate supernatant, and 4 is a lysate precipitant;

FIG. 5 is a SDS-PAGE analysis of codon-optimized pMAL-c5x-PE _ PGRS45 recombinant expression vector, where A is the fusion expression of pMAL-c5x-PE _ PGRS45 in E.coli at 20 ℃, B is the fusion expression of pMAL-c5x-PE _ PGRS45 in E.coli at 37 ℃, M is a protein marker, 1 is E.coli cells before induction, 2 is E.coli cells after induction, 3 is lysate supernatant, and 4 is lysate precipitant;

FIG. 6 is an SDS-PAGE analysis of purified MBP-PE _ PGRS45 fusion proteins;

FIG. 7 is an SDS-PAGE analysis of Factor Xa protease cleavage of MBP;

FIG. 8 shows the indirect ELISA for detecting the titer of polyclonal antibodies.

Detailed Description

If no special requirement exists, the reagent and the strain adopted by the invention are all obtained by conventional purchase of the technicians in the field.

The invention provides a PE _ PGRS45 gene with optimized codons, wherein the nucleotide sequence of the PE _ PGRS45 gene is shown as SEQ ID NO. 1: ATGAGCTTTGTGAATGTGGCCCCGCAGCTGGTTAGTACCGCAGCCGCCGATG CCGCCCGCATTGGTAGCGCTATTAATACCGCAAATACCGCCGCAGCCGCAACC ACCCAGGTGCTGGCTGCAGCCCAGGATGAAGTTAGTACCGCCATTGCCGCAC TGTTTGGCAGTCATGGCCAGCATTATCAGGCAATTAGCGCACAGGTTGCCGC CTATCAGCAGCGCTTTGTTCTGGCCCTGAGTCAGGCCGGTAGTACCTATGCCG TTGCAGAAGCCGCCAGTGCCACCCCGCTGCAGAATGTTCTGGATGCAATTAA TGCCCCGGTGCAGAGTCTGACCGGTCGTCCGCTGATTGGTGACGGCGCCAAT GGTATTGATGGTACCGGCCAGGCAGGCGGTAATGGCGGCTGGCTGTGGGGCA ATGGTGGCAATGGTGGTAGCGGCGCCCCGGGTCAGGCAGGTGGTGCAGGTG GTGCGGCAGGTCTGATTGGTAATGGCGGTGCCGGTGGTGCCGGCGGTCAGG GTTTACCGTTTGAAGCCGGTGCAAATGGCGGCGCCGGTGGCGCAGGTGGTTG GTTATTTGGTAATGGTGGTGCCGGTGGCAATGGCGGTATTGGCGGCGCAGGT ACCAATCTGGCAATTGGTGGTCATGGCGGTAATGGTGGCAACGCCGGCCTGA TTGGCGCCGGTGGTACCGGTGGTGCAGGCGGTACCGGCGGTGGTGAACCGA GTGCAGGCGCCAGTGGTGGCAATGGAGGTAATGGTGGAAATGGTGGTCTGCT GATTGGCAATAGCGGCGATGGTGGCGCCGCAGGCAATGGTGCCGGTATTAGC CAGAATGGCCCGGCAAGTGGTTTTGGTGGCAATGGGGGCCATGCCGGCACC ACCGGTCTGATTGGCAACGGTGGCAATGGTGGAGCAGGTGGTGCTGGCGGT GACGTTAGCGCAGATTTTGGCGGCGTGGGCTTTGGTGGTCAGGGCGGTAATG GAGGTGCAGGTGGCCTGCTGTATGGCAATGGTGGGGCAGGCGGTAACGGTG GTGCCGCAGGTAGCCCGGGCAGTGTGACCGCATTTGGCGGTAATGGGGGTAG CGGTGGCAGCGGTGGTAATGGCGGAAATGCCCTGATTGGCAATGCAGGCGCA GGTGGCAGCGCCGGTGCAGGTGGAAATGGCGCCAGCGCAGGCACCGCCGGT GGTAGCGGTGGTGACGGTGGCAAAGGTGGTAATGGTGGGAGTGTTGGCCTG ATTGGTAACGGTGGCAACGGCGGTAATGGTGGCGCCGGCAGTCTGTTTAATG GCGCACCGGGCTTTGGTGGCCCGGGCGGTAGTGGTGGTGCAAGCCTGCTGG GTCCGCCGGGTCTGGCAGGTACCAACGGTGCCGATGGT are provided. The PE _ PGRS45 gene can be used for obtaining a polyclonal antibody with high titer. Meanwhile, the test results in the specific embodiment of the invention show that the Codon Adaptation Index (CAI) of the gene is increased from 0.73 to 0.90, and the codon adaptation index is improved after the PE _ PGRS45 gene is subjected to codon optimization, so that the codon usage preference and the soluble expression efficiency of the PE _ PGRS45 gene are increased.

The invention provides a recombinant expression vector containing the PE _ PGRS45 gene optimized by the codon, namely pMAL-c5x-PE _ PGRS45, wherein the basic plasmid of the recombinant expression vector comprises pMAL-c5 x; the nucleotide sequence of the gene is located between Nde I and Hind III cleavage sites of pMAL-c5 x. The nucleotide sequence of the gene is preferably inserted into Nde I and Hind III enzyme cutting sites of pMAL-c5x to obtain the recombinant expression vector. The source of pMAL-c5x is not particularly limited in the present invention, and it can be obtained by ordinary purchase by those skilled in the art. The invention adopts pMAL-c5x containing His6-MBP label, can play a role of molecular chaperone, can promote protein to be correctly folded into natural conformation, and improve the solubility level of the protein; MBP also has the effect of resisting proteolysis, can prevent the degradation of chaperone protein, and can improve the expression level and the solubility of expression products. In addition, the pMAL expression vector system contains a histidine tag at the N-terminus, which can facilitate protein folding and affinity purification.

The invention provides a recombinant protein, wherein the sequence of the encoding gene of the recombinant protein comprises a sequence shown in SEQ ID NO.1, namely the sequence of the encoding gene of the recombinant protein is the sequence of the PE _ PGRS45 gene optimized by the codon. In the present invention, the amino acid sequence of the recombinant protein is preferably as shown in SEQ ID NO. 8: MSFVNVAPQLVSTAAADAARIGSAINTANTAAAATTQVLAAAQDEVSTAIAALF GSHGQHYQAISAQVAAYQQRFVLALSQAGSTYAVAEAASATPLQNVLDAINAPV QSLTGRPLIGDGANGIDGTGQAGGNGGWLWGNGGNGGSGAPGQAGGAGGAA GLIGNGGAGGAGGQGLPFEAGANGGAGGAGGWLFGNGGAGGNGGIGGAGTN LAIGGHGGNGGNAGLIGAGGTGGAGGTGGGEPSAGASGGNGGNGGNGGLLIG NSGDGGAAGNGAGISQNGPASGFGGNGGHAGTTGLIGNGGNGGAGGAGGDVS ADFGGVGFGGQGGNGGAGGLLYGNGGAGGNGGAAGSPGSVTAFGGNGGSGG SGGNGGNALIGNAGAGGSAGAGGNGASAGTAGGSGGDGGKGGNGGSVGLIG NGGNGGNGGAGSLFNGAPGFGGPGGSGGASLLGPPGLAGTNGADG are provided.

The invention provides a preparation method of the recombinant expression protein, which comprises the following steps:

after the recombinant expression vector is used for transforming escherichia coli, the recombinant expression vector is sequentially cultured and induced to obtain recombinant protein.

The present invention does not require any particular type of transformation, as is well known in the art. When the method is used for culturing, the adopted culture medium is preferably LB liquid culture medium containing antibiotics; the antibiotic is preferably ampicillin. In the present invention, the LB liquid medium containing ampicillin preferably comprises the following components in concentration: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 50ug/mL of kanamycin. In the invention, the culture temperature is preferably 20-37 ℃, more preferably 25-27 ℃, and more preferably 30-37 ℃; the end criterion of the culture was OD of the bacterial liquid obtained by the culture₆₀₀0.4 to 0.6. The source of the components of the antibiotic-containing LB liquid medium is not particularly limited in the present invention, and those skilled in the art can conveniently purchase the components.

In the induction according to the invention, the culture medium used preferably comprises the following components in the following concentrations: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50ug/mL kanamycin and 0.5mM IPTG; the temperature of the induction is preferably 20-37 ℃, more preferably 25-27 ℃, more preferably 30-37 ℃ and most preferably 37 ℃; the induced rotating speed is preferably 200 r/min; the time for the induction culture is preferably 12 h. The source of the components of the medium is not particularly limited in the present invention, and those skilled in the art can conveniently purchase the medium.

After the induction, the invention preferably further comprises the step of centrifuging and ultrasonically crushing the thalli obtained after the induction to obtain crushed thalli. In the present invention, the time of the centrifugation is preferably 15 min; the centrifugal force of the centrifugation is preferably 8000 g. In the present invention, the efficiency of the ultrasonication is preferably 300W; the mode of ultrasonication is preferably intermittent ultrasonication; the specific mode of the intermittent ultrasonic crushing is preferably work for 8s and intermittent for 8 s; the time of the intermittent ultrasonic crushing is preferably 20 min; the temperature of the ultrasonication is preferably 4 ℃; during the ultrasonic disruption, the thalli obtained after the induction is preferably resuspended in a lysis buffer solution; the lysis buffer preferably comprises 20mM Tris-HCl, 150mM NaCl and 1mM PMSF; the pH of the lysis buffer is preferably 8.0; the sources of the components of the lysis buffer are not particularly limited in the present invention, and may be obtained by routine purchase by those skilled in the art.

After the ultrasonic crushing, the invention preferably further comprises the step of centrifuging the crushed thalli obtained after the ultrasonic crushing to obtain recombinant protein; the centrifugation conditions are preferably 8000g for 15 min.

After the centrifugation, the invention preferably further comprises purifying the recombinant protein obtained from the recombinant protein to obtain a purified recombinant protein; the purification step is not particularly limited in the present invention, and a method known to those skilled in the art may be used. In a particular embodiment of the invention, said purification preferably comprises the following steps: and carrying out first chromatography, cracking and second chromatography on the recombinant protein to obtain the purified recombinant protein (PE _ PGRS45 protein). In the first chromatography and the second chromatography, the Ni-NTA chromatographic column is preferably adopted; the sample injection speed of the chromatography is preferably 0.5 mL/min.

Before the cleavage after the first chromatography, the present invention preferably further comprises washing, eluting and dialyzing the chromatography recombinant protein obtained after the chromatography to remove unbound protein. In the present invention, the washing buffer for washing preferably comprises the following components in concentrations: tris 20mmol/L, imidazole 20mmol/L and NaCl 0.15 mol/L; the pH of the wash buffer is preferably 8.0; the amount of the washing buffer is preferably 100 ml. In the present invention, the elution buffer for elution is preferably an elution buffer, and the elution buffer containing 250mM imidazole preferably comprises the following components in concentration: tris 20mmol/L, imidazole 250mmol/L and NaCl 0.15 mol/L; the pH of the elution buffer containing 250mM imidazole is preferably 8.0; the amount of the washing buffer containing 250mM imidazole is preferably 20 ml. In the present invention, the dialysis is preferably performed in a dialysis bag; the dialysate used for dialysis is preferably Factor Xa protease working fluid; the Factor Xa protease working solution preferably comprises the following concentrations of components 50mM Tris, 1mM CaCl2, and 0.1% Tween-20; the pH value of the Factor Xa protease working solution is preferably 8; the temperature of the dialysis is preferably 4 ℃; the dialysis time is preferably 12 h; the Factor Xa protease working solution is preferably changed for 2 times in the dialysis process; the present invention can remove imidazole by dialysis.

In the present invention, the cleavage is preferably performed under the action of Factor Xa protease; the mass ratio of the Factor Xa protease to the dialyzed protein obtained after dialysis is preferably 1: (10 to 50), more preferably 1: 20; the temperature of the cracking is preferably 4 ℃; the time for the cracking is preferably 36 h; the invention utilizes Factor Xa protease to remove MBP in dialyzed protein (MBP-PE _ PGRS45) in an enzyme digestion mode, and PE _ PGRS45 protein is released.

The invention provides a preparation method of a high-titer polyclonal antibody, which comprises the following steps:

the recombinant protein is used as an antigen immune host to obtain a polyclonal antibody against PE _ PGRS 45; the host is preferably a New Zealand white rabbit.

The method for immunization is not limited in any way, and can be realized by adopting a mode well known by the technical personnel in the field; in particular embodiments of the invention, the method of immunization preferably employs subcutaneous multipoint immunization; the reagent used for immunization preferably comprises a recombinant protein and Freund's incomplete adjuvant; the Freund's incomplete adjuvant preferably comprises liquid paraffin and lanolin; the volume ratio of the paraffin to the wool fat agent is preferably 2: 1; the recombinant protein and Freund's incomplete adjuvant in the reagent for immunization are preferably in equal volume ratio; the frequency of the immunization is preferably 2 weeks/time; the time period for the immunization is preferably 6 weeks.

In the specific embodiment of the invention, indirect ELISA is adopted to detect the titer of the polyclonal antibody, and the result shows that the maximum titer of the polyclonal antibody is 1: 256000. The polyclonal antibody can induce New Zealand white rabbits to generate high titer antibodies, so the polyclonal antibody can be used for detecting and treating tuberculosis.

The invention provides application of the polyclonal antibody prepared by the preparation method in preparing a kit for diagnosing tuberculosis.

The invention provides a kit for diagnosing tuberculosis, which comprises the polyclonal antibody prepared by the preparation method.

In order to further illustrate the present invention, the following detailed descriptions of a codon-optimized gene, a recombinant expression vector, a recombinant protein and a preparation method and application of polyclonal antibody provided by the present invention are provided with reference to the drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

Sequence analysis of PE _ PGRS45

The Mycobacterium tuberculosis PE _ PGRS45 gene has 1386bp nucleotide and codes 39.3kDa amino acid. By performing BLAST sequence alignment analysis in NCBI database (http:// BLAST. NCBI. nlm. nih. gov /), we found that PE _ PGRS45 was mainly present in pathogenic mycobacteria such as M.tuberculosis (M.tuberculosis), M.bovis (M.bovis), M.africana (M.africanum), M.caniti (M.canettii), and M.caprine (M.Caprae) (FIG. 1), suggesting that PE _ PGRS45 may be a virulence factor for M.tuberculosis.

Example 2

PE _ PGRS45 gene codon optimization

Codon optimization has a significant impact on protein translation rate and total protein production, the PE PGRS45 gene has approximately 20% rare codons, as well as multiple tandem or triplet rare codons (fig. 2), which may lead to slow or early termination of translation and thus to low or no protein production. With reference to the gene sequence of Mycobacterium tuberculosis H37Rv strain (GeneID: 888215) PE _ PGRS45(Rv2615c), the PE _ PGRS45 gene was codon optimized by E.coli codon usage analyzer 2.1 (http:// www.faculty.ucr.edu/. about mmadoro/codon/usage. htm) according to the codon preference of Escherichia coli, and synthesized by Nanjing Belge biotechnology Limited under the condition that the amino acid sequence was not changed, to obtain the PE _ PGRS45 gene represented by the nucleotide SEQ ID NO. 1.

Meanwhile, the Codon Adaptation Index (CAI) can be used for evaluating the expression level of the exogenous gene in the host cell, and the optimal range is 0.8-1.0. The gene sequence was optimized using the E.coli codon preference with the assurance that the soluble expression efficiency could be increased by increasing the CAI of PE _ PGRS45 from 0.73 to 0.90 after codon optimization by analysis with an on-line rare codon analysis tool (http:// www.genscript.com/cgi-bin/tools/rare _ codon _ analysis) while ensuring no change in amino acid sequence.

Example 3

Construction of recombinant expression vectors

Cloning the PE _ PGRS45 gene subjected to codon optimization into a pMAL-c5x basic plasmid to obtain a pMAL-c5x-PE _ PGRS45 recombinant expression vector, wherein the cloning process is as follows: the primers used in the PCR cloning procedure are shown in Table 1, and the PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, annealing at 56 deg.C for 15s, extension at 72 deg.C for 1min for 35 cycles, and extension at 72 deg.C for 10 min; and (3) PCR reaction system: template (codon optimized PE _ PGRS45 gene, SEQ ID No. 1): 0.5 μ L, forward primer: 0.5 μ L, downstream primer: 0.5. mu.L, dNTP: 0.5 μ L, DNA polymerase: 0.5. mu.L, 5 × Buffer: 5 μ L, deionized water: 17.5 μ L, total volume: 25 mu L of the solution; carrying out double enzyme digestion on PE _ PGRS45 and pMAL-c5x by using restriction enzymes Nde I and Hind III, wherein the conditions of the double enzyme digestion system are described in Table 4; after recovery of the cleavage products, they were ligated overnight (16 ℃ C., ligation 12h) using T4 DNA ligase, as follows: 2 μ L of pMAL-c5x, 8 μ L of PE _ PGRS45, 1 μ L of T4 DNA Ligase, 2 μ L of 10 XT 4 DNA Ligase Buffer and 7 μ L of ddH₂O, the primers used and the restriction sites are shown in Table 1, and the restriction enzymes are all available from TaKaRa. The ligation product was transformed into Top10 competent cells (Beijing Tiangen Biochemical technology Co., Ltd.), wherein the transformation was performed by the following steps: 1. adding ligation product 5 μ L into Escherichia coli Top10 competent cell 100 μ L, mixing well, and standing on ice for 30 min; heat shock is carried out for 90s at the temperature of 2.42 ℃; 3. immediately placing on ice for 3 min; 4. adding 900 μ L of pre-warmed LB liquid medium; shaking and culturing at 5.37 deg.C and 150rpm/min for 1 h; 6. taking 100 mu L of culture solution, and coating the culture solution on an LB solid culture plate; 7.37 deg.C, fallStanding, and culturing for 16 h.

The positive colonies obtained after the transformation were selected, and double digestion (digestion system is shown in Table 5) and sequencing identification were performed using restriction enzymes Nde I and Hind III, and the sequencing identification results are shown in FIG. 3.

Comparative examples 1 to 1

A recombinant expression vector was constructed in the manner described in example 3, except that the matrix plasmid was pET28a, the restriction enzymes used were Nco I and Xho I, the double enzyme digestion system is described in detail in Table 2, and the resulting recombinant expression vector was pET-28a-PE _ PGRS 45.

Comparative examples 1 to 2

A recombinant expression vector was constructed in the manner described in example 3, except that the matrix plasmid was pET32a, the restriction enzymes used were Nco I and Xho I, the conditions for the two-enzyme digestion system are described in detail in Table 3, and the resulting recombinant expression vector was pET-32a-PE _ PGRS 45.

TABLE 1 primers used for construction of recombinant expression vectors

Note: the single underlined sequence indicates the restriction enzyme site.

TABLE 2 double digestion System and procedure for pET-28a-PE _ PGRS45

Components	Dosage of
		pET-28a-PE_PGRS45	15μL
Nco I	1.5μL
		Xho I	1.5μL
10×K Buffer	5μL
		0.1％BSA	5μL
ddH2O	14μL
		Procedure	Double digestion at 37 deg.C for 12h

TABLE 3 double restriction system and procedure for pET-28a-PE _ PGRS45

Components	Dosage of
		pET-32a-PE_PGRS45	15μL
Nco I	1.5μL
		Xho I	1.5μL
10×K Buffer	5μL
		0.1％BSA	5μL
ddH2O	14μL
		Procedure	Double digestion at 37 deg.C for 12h

TABLE 4 double restriction system and procedure for pET-28a-PE _ PGRS45

Components	Dosage of
		pMAL-c5x-PE_PGRS45	15μL
Nde I	1.5μL
		Hind III	1.5μL
10×K Buffer	5μL
		ddH2O	24μL
Procedure	Double digestion at 37 deg.C for 12h

As shown in FIG. 3, the PE _ PGRS45 gene was cloned into pET-28a, pET-32a and pMAL-c5x vectors, and restriction enzymes Nco I and Xho I were used to perform double restriction on pET-28a-PE _ PGRS45 and pET-32a-PE _ PGRS45 recombinant expression vectors, and restriction enzymes Nde I and Hind III were used to perform double restriction on pMAL-c5x-PE _ PGRS45 recombinant expression vectors, indicating that there is a specific band at about 1500bp position, which is consistent with the expected result. The sequencing result shows that the nucleotide sequence is 100 percent identical to the nucleotide sequence after codon optimization, and proves that the pET-28a-PE _ PGRS45, pET-32a-PE _ PGRS45 and pMAL-c5x-PE _ PGRS45 recombinant expression vectors are successfully constructed.

Example 4

The recombinant expression vector pET-28a-PE _ PGRS45 obtained in comparative example 1-1, the recombinant expression vector pET-32a-PE _ PGRS45 obtained in comparative example 1-2, and the recombinant expression vector pMAL-c5x-PE _ PGRS45 obtained in example 3 were transformed into the expression strain Escherichia coli BL21(DE3) (Beijing Tiangen Biochemical technology Co., Ltd.), positive single colonies were picked up and inoculated into 3mL of LB-containing liquid medium containing antibiotic (wherein the antibiotic used for pET-28a-PE _ PGRS45 was kanamycin resistance, the LB-containing liquid medium was tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, kanamycin 50 ug/mL; the antibiotic used for pET-32a-PE _ PGRS45 and pMAL-c5x-PE _ PGRS45 was ampicillin resistance, the antibiotic-containing LB liquid medium is tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, ampicillin 100ug/mL), and is activated at 37 ℃ overnight.

Respectively inoculating the mixture into 30mL of fresh LB liquid culture medium containing antibiotics according to the volume ratio of 1:100, and performing shake culture until OD₆₀₀0.6. Adding IPTG with final concentration of 0.5mM, shaking for induction culture at 20 deg.C and 37 deg.C at 200r/min for 12h, and inducing protein expression to obtain induced Escherichia coli cell.

Centrifuging at 8000g for 15min, and collecting thallus; then carrying out ultrasonic crushing (300W, work time 8s, pause 8s, total 20min), then centrifuging for 15min under the action of 8000g of centrifugal force, and collecting the induced lysate supernatant and the lysate precipitant.

Then, the supernatant of the lysate, the lysate precipitant, the E.coli cells before induction and the E.coli cells after induction were analyzed by gel electrophoresis detection using 12% SDS-PAGE, and the results are shown in FIGS. 4 and 5.

As can be seen from FIGS. 4 and 5, the fusion tags of the present invention were pET-28a (His6), pET-32a (Trx) and pMAL-c5x (His6-MBP), wherein no significant band was observed in both the supernatant and the pellet of the expression vectors of pET-28a-PE _ PGRS45 and pET-32a-PE _ PGRS45 after ultrasonication (A. about.D in FIG. 4). After IPTG induction of the pMAL-c5x-PE _ PGRS45 expression vector, a distinct specific band (containing the PE _ PGRS45 protein 39kDa and the MBP protein 43kDa) at 82kDa was observed, consistent with the expected protein size (A-B in FIG. 5). It can be seen that neither pET-28a-PE _ PGRS45 nor pET-32a-PE _ PGRS45 can induce the expression of PE _ PGRS45 protein at 20 ℃ or 37 ℃. The recombinant expression vector pMAL-c5x-PE _ PGRS45 can soluble express PE _ PGRS45 protein at 20 ℃ and 37 ℃, and induce the expression level to be higher than 20 ℃ at 37 ℃, and the expression of MBP-PE _ PGRS45 protein is reduced probably because of low temperature. Thus, the pMAL-c5x-PE _ PGRS45 expression vector was selected in subsequent experiments, and expression was induced in large amounts at 37 ℃.

Example 5

The recombinant expression vector pMAL-c5x-PE _ PGRS45 obtained in example 3 contained a histidine tag (6 His-tag) and Maltose Binding Protein (MBP) at the N-terminus, and the recombinant protein obtained in example 4 using this recombinant expression vector was the resulting fusion protein with histidine, and the fusion protein with histidine was purified using a Ni-NTA chromatography column. The method comprises the following specific steps: the cells collected in example 4 were resuspended in lysis buffer (20mM Tris-HCl, 150mM NaCl, 1mM PMSF, pH8.0), sonicated (300w, work 8s, pause 8s, 20min), centrifuged at 8000g for 15min under centrifugal force, the supernatant was collected, and the supernatant was applied to a Ni-NTA column (Novagen) at 0.5 mL/min. Unbound protein was removed by washing with 100mL of washing buffer (Tris 20mmol/L, imidazole 20mmol/L, NaCl 0.15mol/L, pH 8.0). Finally, the MBP-PE _ PGRS45 protein obtained after chromatography was eluted with 20ml of an elution buffer containing 250mM imidazole (Tris 20mmol/L, imidazole 250mmol/L, NaCl 0.15mol/L, pH 8.0). The purified protein was transferred to a dialysis bag, dialyzed overnight at 4 ℃ to remove imidazole, and subjected to SDS-PAGE on MBP-PE _ PGRS45 protein.

The electrophoresis result is shown in FIG. 6, and the purity of the purified MBP-PE _ PGRS45 protein is 91%.

Example 5

MBP-PE _ PGRS45 protein cleavage and purification

The pMAL-c5x base plasmid contains a Factor Xa protease site between the fusion protein MBP and the PE _ PGRS45 protein. And (3) incubating the purified MBP-PE _ PGRS45 protein and Factor Xa protease, and loading the MBP-PE _ PGRS45 protein after enzyme digestion to a Ni-NTA chromatographic column. Because the N end of the MBP protein has a histidine tag, the removed MBP protein is combined with a Ni-NTA chromatographic column, and the effluent protein is PE _ PGRS45 protein (recombinant protein) after the removal of the MBP protein, and the specific steps are as follows:

the purified MBP-PE _ PGRS45 protein obtained in example 4 was placed in dialysis bags in Factor Xa protease working solution (50mM Tris, 1mM CaCl)₂0.1% Tween-20, pH8.0) overnight (12h), during which 2 changes of Factor Xa protease working fluid were made. And (3) adding Factor Xa protease after dialysis, wherein the mass ratio of the Factor Xa protease to dialyzed protein obtained after dialysis is 1: incubating for 36h at 20, 4 ℃ to obtain a lysis mixture. And (2) passing the cleavage mixture through a Ni-NTA chromatographic column, wherein the Factor Xa protease can cleave the MBP-PE _ PGRS45 enzyme into two parts of MBP and PE _ PGRS45, the N end of the MBP contains a histidine tag and can be combined with the Ni-NTA chromatographic column, and the PE _ PGRS45 does not contain the histidine tag and can flow out, namely PE _ PGRS45 protein except the MBP, so that the effluent is PE _ PGRS45 protein without the MBP, namely recombinant protein, and performing SDS-PAGE electrophoresis on the recombinant protein.

The electrophoresis result is shown in FIG. 7, lane 1 shows that the Ni-NTA chromatographic column after digestion adsorbs protein, and has an obvious specific band at about 43KD, which is consistent with the expected size of MBP protein, and lane 2 shows that the effluent after digestion has an obvious specific band at about 39kDa, which is consistent with the expected size of PE _ PGRS45 protein. The MBP-PE _ PGRS45 protein MBP is successfully cut off, and the purified PE _ PGRS45 protein band is single, so that the requirements of subsequent experiments are met.

Example 6

Preparation and identification of PE _ PGRS45 polyclonal antibody

400 mu g of the purified PE _ PGRS45 protein obtained in example 5 was mixed with an equal volume of Freund's incomplete adjuvant (paraffin: lanolin agent: 2:1), and the New Zealand white rabbits were immunized subcutaneously and multiply for 6 weeks, 4 times, 3 times, and the injection amount was 400. mu.g/mouse. Taking the serum before immunization as a negative control, and measuring the serum titer by using an indirect ELISA method, the specific operation is as follows:

(1) antigen coating: the PE _ PGRS45 protein (5. mu.g/mL) was coated in a 96-well plate at 100. mu.L/well and left overnight at 4 ℃. Discard the internal solution, wash with PBST 3 times, seal with 5% skimmed milk powder at 37 deg.C for 1 h.

(2) The polyclonal antibody PE _ PGRS45 (0.12mg/mL) was diluted to 0.12. mu.g/mL with PBS, started at 1:1000, diluted at a double rate to 1:512000, added to the wells of step (1) at 100. mu.L/well and incubated at 37 ℃ for 1 h. Discarding the internal solution, washing with PBST for 3 times, adding HRP-labeled goat anti-rabbit IgG (1:5000) at 100 μ L/well, incubating at 37 deg.C for 1 h; adding TMB color development solution, 100 μ L/hole, keeping away from light at 37 deg.C for 15min, and detecting A450nm absorbance value with microplate reader. The indirect ELISA test results are shown in FIG. 8, and the maximum titer of the polyclonal antibody was determined to be 1: 256000.

As can be seen from the above, the nucleotide sequence provided by the invention is the PE _ PGRS45 gene shown in SEQ ID NO. 1. The PE _ PGRS45 gene can be used for obtaining a polyclonal antibody with high titer. The experimental results in the specific examples of the present invention show that the Codon Adaptation Index (CAI) of the gene increases from 0.73 to 0.90.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Bengbu institute of medicine

<120> preparation methods and applications of codon optimized gene, recombinant expression vector, recombinant protein and polyclonal antibody

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1383

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgagctttg tgaatgtggc cccgcagctg gttagtaccg cagccgccga tgccgcccgc 60

attggtagcg ctattaatac cgcaaatacc gccgcagccg caaccaccca ggtgctggct 120

gcagcccagg atgaagttag taccgccatt gccgcactgt ttggcagtca tggccagcat 180

tatcaggcaa ttagcgcaca ggttgccgcc tatcagcagc gctttgttct ggccctgagt 240

caggccggta gtacctatgc cgttgcagaa gccgccagtg ccaccccgct gcagaatgtt 300

ctggatgcaa ttaatgcccc ggtgcagagt ctgaccggtc gtccgctgat tggtgacggc 360

gccaatggta ttgatggtac cggccaggca ggcggtaatg gcggctggct gtggggcaat 420

ggtggcaatg gtggtagcgg cgccccgggt caggcaggtg gtgcaggtgg tgcggcaggt 480

ctgattggta atggcggtgc cggtggtgcc ggcggtcagg gtttaccgtt tgaagccggt 540

gcaaatggcg gcgccggtgg cgcaggtggt tggttatttg gtaatggtgg tgccggtggc 600

aatggcggta ttggcggcgc aggtaccaat ctggcaattg gtggtcatgg cggtaatggt 660

ggcaacgccg gcctgattgg cgccggtggt accggtggtg caggcggtac cggcggtggt 720

gaaccgagtg caggcgccag tggtggcaat ggaggtaatg gtggaaatgg tggtctgctg 780

attggcaata gcggcgatgg tggcgccgca ggcaatggtg ccggtattag ccagaatggc 840

ccggcaagtg gttttggtgg caatgggggc catgccggca ccaccggtct gattggcaac 900

ggtggcaatg gtggagcagg tggtgctggc ggtgacgtta gcgcagattt tggcggcgtg 960

ggctttggtg gtcagggcgg taatggaggt gcaggtggcc tgctgtatgg caatggtggg 1020

gcaggcggta acggtggtgc cgcaggtagc ccgggcagtg tgaccgcatt tggcggtaat 1080

gggggtagcg gtggcagcgg tggtaatggc ggaaatgccc tgattggcaa tgcaggcgca 1140

ggtggcagcg ccggtgcagg tggaaatggc gccagcgcag gcaccgccgg tggtagcggt 1200

ggtgacggtg gcaaaggtgg taatggtggg agtgttggcc tgattggtaa cggtggcaac 1260

ggcggtaatg gtggcgccgg cagtctgttt aatggcgcac cgggctttgg tggcccgggc 1320

ggtagtggtg gtgcaagcct gctgggtccg ccgggtctgg caggtaccaa cggtgccgat 1380

ggt 1383

<210> 2

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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catgccatgg atgagctttg tgaatgtggc c 31

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

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ccgctcgagt taaccatcgg caccgttggt acc 33

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

catgccatgg atgagctttg tgaatgtggc c 31

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ccgctcgagt taaccatcgg caccgttggt acc 33

<210> 6

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<213> Artificial Sequence (Artificial Sequence)

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ggaattcata tgagctttgt gaatgtggcc 30

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<213> Artificial Sequence (Artificial Sequence)

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cccaagcttt taaccatcgg caccgttggt acc 33

<210> 8

<211> 461

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ser Phe Val Asn Val Ala Pro Gln Leu Val Ser Thr Ala Ala Ala

1 5 10 15

Asp Ala Ala Arg Ile Gly Ser Ala Ile Asn Thr Ala Asn Thr Ala Ala

20 25 30

Ala Ala Thr Thr Gln Val Leu Ala Ala Ala Gln Asp Glu Val Ser Thr

35 40 45

Ala Ile Ala Ala Leu Phe Gly Ser His Gly Gln His Tyr Gln Ala Ile

50 55 60

Ser Ala Gln Val Ala Ala Tyr Gln Gln Arg Phe Val Leu Ala Leu Ser

65 70 75 80

Gln Ala Gly Ser Thr Tyr Ala Val Ala Glu Ala Ala Ser Ala Thr Pro

85 90 95

Leu Gln Asn Val Leu Asp Ala Ile Asn Ala Pro Val Gln Ser Leu Thr

100 105 110

Gly Arg Pro Leu Ile Gly Asp Gly Ala Asn Gly Ile Asp Gly Thr Gly

115 120 125

Gln Ala Gly Gly Asn Gly Gly Trp Leu Trp Gly Asn Gly Gly Asn Gly

130 135 140

Gly Ser Gly Ala Pro Gly Gln Ala Gly Gly Ala Gly Gly Ala Ala Gly

145 150 155 160

Leu Ile Gly Asn Gly Gly Ala Gly Gly Ala Gly Gly Gln Gly Leu Pro

165 170 175

Phe Glu Ala Gly Ala Asn Gly Gly Ala Gly Gly Ala Gly Gly Trp Leu

180 185 190

Phe Gly Asn Gly Gly Ala Gly Gly Asn Gly Gly Ile Gly Gly Ala Gly

195 200 205

Thr Asn Leu Ala Ile Gly Gly His Gly Gly Asn Gly Gly Asn Ala Gly

210 215 220

Leu Ile Gly Ala Gly Gly Thr Gly Gly Ala Gly Gly Thr Gly Gly Gly

225 230 235 240

Glu Pro Ser Ala Gly Ala Ser Gly Gly Asn Gly Gly Asn Gly Gly Asn

245 250 255

Gly Gly Leu Leu Ile Gly Asn Ser Gly Asp Gly Gly Ala Ala Gly Asn

260 265 270

Gly Ala Gly Ile Ser Gln Asn Gly Pro Ala Ser Gly Phe Gly Gly Asn

275 280 285

Gly Gly His Ala Gly Thr Thr Gly Leu Ile Gly Asn Gly Gly Asn Gly

290 295 300

Gly Ala Gly Gly Ala Gly Gly Asp Val Ser Ala Asp Phe Gly Gly Val

305 310 315 320

Gly Phe Gly Gly Gln Gly Gly Asn Gly Gly Ala Gly Gly Leu Leu Tyr

325 330 335

Gly Asn Gly Gly Ala Gly Gly Asn Gly Gly Ala Ala Gly Ser Pro Gly

340 345 350

Ser Val Thr Ala Phe Gly Gly Asn Gly Gly Ser Gly Gly Ser Gly Gly

355 360 365

Asn Gly Gly Asn Ala Leu Ile Gly Asn Ala Gly Ala Gly Gly Ser Ala

370 375 380

Gly Ala Gly Gly Asn Gly Ala Ser Ala Gly Thr Ala Gly Gly Ser Gly

385 390 395 400

Gly Asp Gly Gly Lys Gly Gly Asn Gly Gly Ser Val Gly Leu Ile Gly

405 410 415

Asn Gly Gly Asn Gly Gly Asn Gly Gly Ala Gly Ser Leu Phe Asn Gly

420 425 430

Ala Pro Gly Phe Gly Gly Pro Gly Gly Ser Gly Gly Ala Ser Leu Leu

435 440 445

Gly Pro Pro Gly Leu Ala Gly Thr Asn Gly Ala Asp Gly

450 455 460