CN115724941A - Gamma-interferon recombinant antigen and application thereof - Google Patents
Gamma-interferon recombinant antigen and application thereof Download PDFInfo
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Abstract
The invention relates to the fields of biomedicine and in-vitro diagnosis, in particular to a gamma-interferon recombinant antigen and application thereof. The gamma-interferon recombinant antigen is obtained by protein combination and antigen epitope screening, and has an amino acid sequence shown as SEQ ID NO. 11, and experimental results show that the gamma-interferon recombinant antigen with the amino acid shown as SEQ ID NO. 11 has high expression quantity, strong antigenicity and high stability, can fully expose dominant epitopes in animal immunity, and has higher immunological activity.
Description
Technical Field
The invention relates to the fields of biomedicine and in-vitro diagnosis, in particular to a gamma-interferon recombinant antigen and application thereof.
Background
As early as 30 years of the last century, it has been found that the body, after being infected with a certain virus, interferes with another virus. Until 1957, the british scientist Isaacs and swiss scientist Lindenman, in studying the interference phenomenon of influenza virus, discovered a soluble molecule that could interfere with the viral infection, named interferon, IFN, chinese translation to interferon. Interferons (IFNs) are a family of multifunctional cytokines with broad-spectrum antiviral, antiproliferative and immunomodulatory activity, which can be classified into type i (including IFN- α and β), type ii (IFN- γ) and type III (IFN- λ) depending on the binding receptor, among which the α -IFN plays an important role in the control of viral infections in the body.
IFN-gamma is a pleiotropic cytokine that can reverse the host cell during immune responses, ranging from chronic inflammatory cell transformation, monocytes/macrophages, to antitumor, cytotoxic T Cells (CTL), th1, NK, NKT cells. IFN-gamma has an anti-tumor effect in anti-tumor immune response, but normal cells can also promote malignant transformation after being exposed to the IFN-gamma for a long time, and the IFN-gamma is the most important marker for diagnosing Tuberculosis (TB) at present; at present, a double-antibody sandwich enzyme-linked immunosorbent assay technology is adopted to monitor the content of IFN-gamma in real time, and the IFN-gamma can be found in time when the content in a human body is abnormal. The IFN-gamma protein has the full length of 166AA, more alpha helical regions exist, the exposure of epitopes is not facilitated, and the mature IFN-gamma protein is in a dimer form, so that dominant epitopes are difficult to expose on the surface of the mature IFN-gamma protein. The commercial recombinant human IFN-gamma protein is expensive in price, mostly is an injection preparation, and is not suitable for animal immunization. The natural human IFN-gamma protein is difficult to extract and difficult to produce in quantity. Therefore, the development of the recombinant human IFN-gamma protein which has lower cost and can expose the dominant epitope has important significance for developing anti-human IFN-gamma antibodies.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a recombinant gamma-interferon antigen and its application.
The invention provides a gamma-interferon recombinant protein, the amino acid sequence of which is shown as SEQ ID NO: shown at 11.
According to the protein structure and dominant epitope analysis of the gamma-interferon with UNIPORT accession number P01579, the invention screens out short-length gamma-interferon recombinant proteins IA1, IA2 and IA3, wherein the gamma-interferon recombinant protein IA1 has the amino acid sequence shown in SEQ ID NO:11, or a pharmaceutically acceptable salt thereof. The result of antigen immunocompetence identification shows that the gamma-interferon recombinant protein IA1 has high expression, strong antigenicity and high stability, and the effect of the gamma-interferon recombinant protein IA1 in animal immunity is superior to that of other antigens.
The invention provides a nucleic acid for coding the gamma-interferon recombinant protein, which has the nucleotide sequence shown as SEQ ID NO: 2.
In the present invention, the nucleic acid encoding the recombinant protein of interferon-gamma is codon-optimized for E.coli, and these optimizations include but are not limited to: codon usage bias, elimination of secondary structures that are detrimental to expression (such as hairpin structures), alteration of GC content, cpG dinucleotide content, secondary structure of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA instability regions, repetitive sequences (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning.
In the present invention, the nucleic acid may be DNA, RNA, cDNA or PNA. In embodiments of the invention, the nucleic acid is in the form of DNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. Nucleic acids may include nucleotide sequences with different functions, such as coding regions and non-coding regions such as regulatory sequences (e.g., promoters or transcription terminators). Nucleic acids can be linear or circular in topology. The nucleic acid may be, for example, part of a vector, such as an expression or cloning vector, or a fragment. The nucleic acids may be obtained directly from natural sources or may be prepared with the aid of recombinant, enzymatic or chemical techniques.
The invention provides an expression module of the gamma-interferon recombinant protein, which comprises at least one of a promoter, a terminator and the nucleic acid.
In the present invention, the expression module further includes one or more expression modules formed by combining the nucleic acids of the present invention in tandem, fusion expression, or other feasible manners, which is not limited in the present invention.
The invention also provides a transcription unit of the gamma-interferon recombinant protein, wherein the transcription unit refers to a DNA sequence from a promoter to a terminator. The promoter and the terminator can also comprise a regulatory fragment or a fusion protein on two sides or between the promoter and the terminator; the regulatory fragment may include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site, transmembrane signal peptide, and homologous recombination site, such as f1 origin, lac operator signal peptide, etc., operably linked to a nucleic acid sequence; the fusion protein includes but is not limited to TrxA or mbp.
The invention provides a recombinant vector comprising a vector backbone and a nucleic acid according to the invention.
Furthermore, the source of the vector of the present invention includes plant, animal, microorganism or virus vector, which is not limited in this respect. The microbial carrier source includes but is not limited to prokaryotic microorganisms, eukaryotic microorganisms; the prokaryotic microorganism includes but is not limited to Escherichia coli; the eukaryotic microorganism includes but is not limited to yeast. The prokaryotic microorganism vector includes but is not limited to pET32a, pET28a, or pCold I. In some specific embodiments, the vector backbone is pET32a.
The recombinant vector of the present invention, which refers to a nucleic acid vector, is a recombinant DNA molecule comprising the desired coding sequence and appropriate nucleic acid sequences or elements necessary for expression of the operably linked coding gene in a particular host organism. Nucleic acid sequences or elements necessary for expression in bacteria include promoters, ribosome binding sites and possibly other sequences. Bacterial cells are known to utilize promoters, enhancers, and terminators. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. In the present specification, "plasmid" and "vector" may sometimes be used interchangeably, since plasmids are the most commonly used form of vector at present. However, the present invention is intended to include such other forms of expression vectors which serve equivalent functions, which are or will become known in the art, including, but not limited to: plasmids, phage particles, viral vectors and/or simply potential genomic inserts.
The invention provides a host which comprises any one or more of the following I) to II):
i) Chromosomally integrating a nucleic acid according to the invention;
II), transfection or transformation of a recombinant vector according to the invention.
Further, the host of the present invention includes, but is not limited to, bacteria, fungi, viruses, plant cells or mammalian cells, and the present invention is not limited thereto. Further, in some embodiments, the present invention uses escherichia coli as a host, specifically, escherichia coli BL21. The present invention uses vectors constructed using recombinant DNA techniques to transform or transfect host cells such that the transformed host cells have the ability to replicate the vector encoding the protein or express the desired protein.
In the present invention, the method for transformation comprises: chemical conversion and electrical conversion; the transfection method comprises calcium phosphate coprecipitation, an artificial liposome method and virus transfection. The virus transfection comprises adenovirus transfection, adeno-associated virus transfection, lentivirus transfection and the like. In some specific embodiments, the present invention performs the construction of the host by chemical transformation.
The invention provides a preparation method of the gamma-interferon recombinant protein, which is to culture a host according to the invention to obtain a culture containing the gamma-interferon recombinant protein.
The application of any one of the following i) to v) in preparing a gamma-interferon antibody or a gamma-interferon detection product:
i) The gamma-interferon recombinant protein provided by the invention;
ii) a nucleic acid according to the invention;
iii) The recombinant vector of the invention;
iv) a host according to the invention;
v) culture containing the gamma-interferon recombinant protein prepared by the preparation method.
The invention provides a gamma-interferon antibody, which is prepared by taking the gamma-interferon recombinant protein as an antigen.
The invention provides a preparation method of a gamma-interferon antibody, which is characterized in that after an animal is immunized by using the gamma-interferon recombinant protein as an antigen, blood is collected, separated and purified to obtain the gamma-interferon antibody.
Animals described in the present invention include, but are not limited to, rabbits, mice, sheep, chickens, etc.
The invention provides a gamma-interferon detection product, which comprises the gamma-interferon antibody.
The gamma-interferon detection product can be applied to detection of content change of gamma-interferon caused by tuberculosis, immunodeficiency diseases, malignant tumors, aplastic anemia and the like.
Further, the gamma-interferon detection product comprises a kit, which comprises a buffer solution and the gamma-interferon antibody.
The invention provides a method for detecting gamma-interferon, which is used for detecting gamma-interferon by using a gamma-interferon detection product or a kit.
The gamma-interferon recombinant antigen is obtained by protein combination and antigen epitope screening, and has an amino acid sequence shown as SEQ ID NO. 11, and experimental results show that the gamma-interferon recombinant antigen with the amino acid shown as SEQ ID NO. 11 has high expression quantity, strong antigenicity and high stability, can fully expose dominant epitopes in animal immunity, and has higher immunological activity.
Drawings
FIG. 1 shows identification charts of gene amplification of IA1, IA2 and IA 3;
FIG. 2 shows the identification of IA1 and IA2 SDS-PAGE, in which 1#, 2#, and 3# represent selected clones;
FIG. 3 shows an IA3 SDS-PAGE electrophoresis, wherein # 1, # 2 and # 3 represent selected clones;
FIG. 4 shows the results of Western Blot of IA1, IA2, and IA3 with commercial antibodies, IA1 reacted with commercial anti-interferon-gamma antibodies.
Detailed Description
The invention provides a gamma-interferon recombinant antigen and application thereof, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the purpose. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The nucleotide sequence of the human gamma-interferon gene recombinant protein is as follows: <xnotran> atgcaagatccgtatgtgaaagaagcggaaaacctgaaaaaatattttaacgcgggccatagcgatgtggcggataacggcaccctgtttctgggcattctgaaaaactggaaagaagaaagcgatcgcaaaattatgcagagtcagattgtgagcttttattttaaactgtttaaaaactttaaagatgatcagagcattcagaaaagcgttgaaacgattaaagaagatatgaacgtgaagtttttcaacagtaacaaaaagaaacgcgatgattttgaaaaactgaccaactatagcgtgaccgatctgaacgtgcagcgcaaagcgattcatgaactgattcaagtgatggcggaactgagcccggcggcgaaaaccggcaaacgcaaacgcagtcagatgctgtttcgcggctaa ( SEQ ID NO:1 ); </xnotran>
The nucleotide sequence of the human gamma-interferon gene truncated IA1 recombinant protein is as follows: <xnotran> atgcaagatccgtatgtgaaagaagcggaaaacctgaaaaaatattttaacgcgggccatagcgatgtggcggataac ggcaccctgtttctgggcattctgaaaaactggaaagaagaaagcgatcgcaaaattatgcagagtcagattgtgtaa ( SEQ ID NO:2 ); </xnotran>
The nucleotide sequence of the human gamma-interferon gene truncation IA2 recombinant protein is as follows: <xnotran> cagagtcagattgtgagcttttattttaaactgtttaaaaactttaaagatgatcagagcattcagaaaagcgttgaaacga ttaaagaagatatgaacgtgaagtttttcaacagtaacaaaaagaaacgcgattaa ( SEQ ID NO:3 ); </xnotran>
The nucleotide sequence of the human gamma-interferon gene truncated IA3 recombinant protein is as follows: <xnotran> aaaaagaaacgcgatgattttgaaaaactgaccaactatagcgtgaccgatctgaacgtgcagcgcaaagcgattcat gaactgattcaagtgatggcggaactgagcccggcggcgaaaaccggcaaacgcaaacgcagtcagatgctgtttc gcggctaa ( SEQ ID NO:4 ); </xnotran>
The nucleotide sequence of the upstream primer IA1-F is as follows: cggggatccatgcaagatcgtatgtgaaag (shown in SEQ ID NO: 5);
the nucleotide sequence of the downstream primer IA1-R is as follows: ccgctcgagttacacaatctgactgcataa (as shown in SEQ ID NO: 6);
the nucleotide sequence of the upstream primer IA2-F is as follows: cggggatcccagtcagattgtgagcttta (shown as SEQ ID NO: 7);
the nucleotide sequences of the downstream primers IA2-R5 are as follows: ccgctcgagtttaatcgcgtttttttttgttactgttga (shown in SEQ ID NO: 8);
the nucleotide sequence of the upstream primer IA3-F is as follows: cggggatccaaaaaaagaacgcgatgatttgaa (shown in SEQ ID NO: 9);
the nucleotide sequence of the downstream primer IA3-R is as follows: ccgctcgagttagccgcgaaaacagctgactgcg (shown as SEQ ID NO: 10);
the amino acid sequence of the gamma-interferon recombinant protein IA1 is as follows: MQDPYVKEALKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQI V (shown in SEQ ID NO: 11).
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 human Gamma-Interferon IA1 antigen Gene recombinant protein
Reagents such as Prime STAR PCR Mix, DL2000Marker, DL5000Marker, ligation Mix ligation kit, bamHI, XBaI restriction enzymes, IPTG, etc. were purchased from precious bioengineering (da lian) ltd. Plasmid extraction kits, DNA gel recovery kits, and the like were purchased from OMEGA.
1. Chemical Synthesis of DNA fragments of interest
Human gamma-interferon synthesized by using Jinweizhi gene is used as DNA template.
2. PCR amplification
1. Primer design
According to the gamma-interferon whole gene sequence with UNIPORT accession number P01579 and codon optimization of the gene sequence aiming at escherichia coli, a specific primer for amplifying a gamma-interferon IA1 antigen gene DNA fragment is designed according to the optimized DNA sequence, double enzyme cutting sites of BamH I and X baI are introduced into the tail ends of an upstream primer and a downstream primer, and the length of an expected amplification fragment is about 156bp. Specific primers designed to amplify the a antigen gene were as follows:
upstream primers IA1-F:5' cggggattcaagatcctgtatgtgaaaag (shown as SEQ ID NO: 5);
downstream primers IA1-R:5' ccgctccgagttacacaaaatctgactgctgatctataa-;
and (3) PCR amplification: the gamma-interferon whole gene sequence DNA is used as a template, and a PCR amplification product of the A antigen is obtained by utilizing a PCR method for amplification through a designed specific primer.
The PCR reaction system is as follows: 20 μ L system: mg (magnesium) 2+ 1.5mmol/L, dNTP 0.2mmol/L, taq enzyme 1U, primer 5pmol/L, sample DNA 100-200 ng, and the balance of ultrapure water.
The PCR reaction program is: 10min at 95 ℃; 10 cycles of 94 ℃ 20s,63 ℃ 30s and 72 ℃ lmin; performing 25 cycles at 94 ℃ for 20s,60 ℃ for 30s and 72 ℃ for 1 min; extending for 5min at 72 ℃; storing at 4 ℃.
2. Detection of PCR products
And (3) identifying the amplified fragment by electrophoresis, detecting the amplified product on 1% agarose gel, wherein the voltage is 10v/cm, and the electrophoresis is performed for about 20 min. The length of the desired fragment was marked using the DNA molecular marker DL2000 (TAKARA, dalianbao Biopsies), and the results are shown in FIG. 1. As can be seen from FIG. 1, the amplified fragment was 156bp, which is in agreement with the expectation.
3. Recovery and purification of PCR-specific fragments
Use of
Gel Extraction Kit (Omega Co., ltd.)And recovering and purifying the amplification product.
3. Construction of recombinant expression vectors
1. Construction of recombinant plasmid pET32a-IA1
Purifying and recovering the correctly identified PCR specific fragment according to the DNA gel recovery kit specification, and identifying the gel recovery products BamHI and Xba I by double enzyme digestion, wherein the enzyme digestion system comprises: 10 XBuffer 1. Mu.l, bamHI 0.5. Mu.l, xbaI 0.5. Mu.l, recombinant plasmid 8. Mu.l. The enzyme was digested at 37 ℃ for 1.5h, and the product of the digestion was identified by electrophoresis on 1% agarose.
2. Construction of recombinant expression vector pET32a-IA1
And carrying out double enzyme digestion on the PCR specific fragment and the expression vector PET32A with BamH I and Xba I at the same time, and connecting to obtain a recombinant expression vector pET32A-IA1.
Connecting a reaction system: 5.5 ul of the target gene fragment, 2 ul of the expression vector, and 2 XT 4DNA Ligase Mix7.5 ul. Flash separation, mix well and ligate overnight at 16 ℃. To obtain a ligation product-recombinant plasmid pET32a-IA1.
Transferring 5 mu L of recombinant plasmid pET32a-IA1 into DH5a competent cells, slowly blowing and uniformly mixing, and standing on ice for 30min; performing heat shock at 42 ℃ for 90s, performing ice bath for 2min, adding 445 mu L of LB liquid culture medium, performing shaking culture at 37 ℃ at 200-250 r/min for 1h, performing centrifugation at 4000r/min for 2min, and mixing uniformly; uniformly coating the bacterial liquid on an LB agar plate containing antibiotics, and placing the bacterial liquid in a thermostat at 37 ℃ after the bacterial liquid is absorbed for overnight culture until a single bacterial colony appears; selecting bacteria, and culturing overnight at 37 ℃ under shaking at 300 r/min.
PCR screening positive clone of bacterial liquid: and (3) amplifying the IA1 gene by PCR by using the turbid bacterial liquid as a template. The product was checked for amplification by electrophoresis on a 1% agarose gel.
And (3) identification result: the IA1 gene is cloned to a pET32a vector, expected bands are obtained after PCR and enzyme digestion identification, and the length of IA1 in the recombinant plasmid pET32a-IA1 is 156bp. The sequencing result of pET32a-IA1 is consistent with the expectation through the optimized gamma-interferon sequence, and the success of the construction of the recombinant expression vector is shown.
4. Transformation of
Transforming the recombinant expression vector pET32a-IA1 into E coli BL21 expression host bacteria, and screening positive expression bacteria
5. Inducible expression
Inoculating the screened positive expression strain containing the recombinant expression vector pET32a-IA1 into an LB culture medium, culturing overnight at 37 ℃, carrying out amplification culture according to a ratio of 1.
6. Purification of
Centrifuging the bacteria liquid after induction expression for 10min at 12000r/min, enriching thalli, resuspending the thalli by using 20mmol/L Tris-HCl buffer solution with the pH value of 8.0 according to the volume ratio of 1.
Example 2 human Gamma-Interferon IA2 antigen Gene recombinant protein
1. Chemical Synthesis of DNA fragment of interest
Human gamma-interferon synthesized by using Jinweizhi gene is used as DNA template.
2. PCR amplification
1. Primer design
According to the gamma-interferon whole gene sequence with UNIPORT accession number P01579 and codon optimization of the gene sequence aiming at escherichia coli, a specific primer for amplifying a gamma-interferon IA2 antigen gene DNA fragment is designed according to the optimized DNA sequence, double enzyme cutting sites of BamHI and XbaI are introduced into the tail ends of an upstream primer and a downstream primer, and the length of an expected amplification fragment is about 138bp. Specific primers designed to amplify the a antigen gene were as follows:
the upstream primer IA2-F:5 'cggggatccagtcagtcaattgtgagcttta-3' (shown as SEQ ID NO: 7);
downstream primers IA2-R:5 'ccgctccgagttaaatccgtgttttttttgttactgttga-3' (shown as SEQ ID NO: 8);
and (3) PCR amplification: and (3) amplifying by using a PCR method by using DNA as a template and a designed specific primer to obtain an A antigen PCR amplification product.
The PCR reaction system is as follows: 20 μ L system: mg (magnesium) 2+ 1.5mmol/L, dNTP 0.2mmol/L, taq enzyme 1U, primers each 5pmol/L, sample DNA 100-200 ng, and the balance of ultrapure water.
The PCR reaction program is: 10min at 95 ℃; 10 cycles of 94 ℃ 20s,63 ℃ 30s and 72 ℃ lmin; performing 25 cycles at 94 ℃ for 20s,60 ℃ for 30s and 72 ℃ for 1 min; extending for 5min at 72 ℃; storing at 4 ℃.
2. Detection of PCR products
And (3) identifying the amplified fragment by electrophoresis, detecting the amplified product on 1% agarose gel, wherein the voltage is 10v/cm, and the electrophoresis is performed for about 20 min. The length of the target fragment was marked using the DNA molecular marker DL2000 (TAKARA, dalianbao Biometrics), and as can be seen in FIG. 1, the IA2 amplified fragment was 138bp, which is in agreement with the expectation.
3. Recovery and purification of PCR-specific fragments
3. Construction of recombinant expression vectors
1. Construction of recombinant plasmid pET32a-IA2
Purifying and recovering the products of the PCR specific fragments which are identified correctly according to the instruction of the DNA gel recovery kit, and carrying out enzyme digestion on recombinant plasmids of the gel recovery products: extracting the plasmid according to the operation steps of a plasmid extraction kit (OMEGA), and performing double enzyme digestion identification by using BamH I and XbaI, wherein the enzyme digestion system comprises the following steps: 10 XBuffer 1. Mu.l, bamHI 0.5. Mu.l, xbaI 0.5. Mu.l, recombinant plasmid 8. Mu.l. The enzyme was cleaved at 37 ℃ for 1.5h.
2. Construction of recombinant expression vector pET32a-IA2
And carrying out double enzyme digestion on the PCR specific fragment and the expression vector pET32a by BamH I and XbaI, and connecting to prepare a recombinant expression vector pET32a-IA2.
Connecting a reaction system: 5.5 ul of the target gene fragment, 2 ul of the expression vector, and 2 XT 4DNA Ligase Mix7.5 ul. Flash separation, mix well and ligate overnight at 16 ℃. Obtaining a ligation product-recombinant plasmid pET32a-IA2
Transferring 5 mu L of recombinant plasmid pET32a-IA2 into DH5 alpha competent cells, slowly blowing and uniformly mixing, and standing on ice for 30min; thermally shocking at 42 deg.C for 90s, ice-cooling for 2min, adding 445 μ L LB liquid culture medium, shake-culturing at 37 deg.C for 1h at 200-250 r/min, centrifuging at 4000r/min for 2min, and mixing; uniformly coating the bacterial liquid on an LB agar plate containing antibiotics, and placing the bacterial liquid in a thermostat at 37 ℃ after the bacterial liquid is absorbed for overnight culture until a single bacterial colony appears; selecting bacteria, and culturing overnight at 37 ℃ under shaking at 300 r/min.
PCR screening positive clone of bacterial liquid: and (3) carrying out PCR amplification on the IA2 gene by using turbid bacterial liquid as a template. The product was checked for amplification by electrophoresis on a 1% agarose gel.
And (3) identification result: the IA2 gene is cloned to a pET32a vector, expected strips are obtained after PCR and enzyme digestion identification, and the sequencing result of the pET32a-IA2 is consistent with the optimized sequence of the gamma-interferon IA2, which indicates that the construction of the recombinant expression vector is successful.
4. Transformation of
Transforming the recombinant expression vector pET32a-IA2 into E.coli.BL21 expression host bacteria, and screening positive expression bacteria
5. Inducible expression
Inoculating the screened positive expression strain containing the recombinant expression vector pET32a-IA2 into an LB culture medium, culturing overnight at 37 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) into the bacterial liquid to a final concentration of 0.4mmol/L when the bacterial liquid OD600nm =0.6 is subjected to amplification culture according to a ratio of 1.
6. Purification of
Centrifuging the bacteria solution after induced expression for 10min at 12000r/min, enriching thalli, resuspending the thalli by using 20mmol/L Tris-HCl buffer solution with the pH value of 8.0 according to the volume ratio of 1. Example 3 human Gamma-Interferon IA3 antigen Gene recombinant protein
1. Chemical Synthesis of DNA fragments of interest
Human gamma-interferon synthesized by using Jinweizhi gene is used as DNA template.
2. PCR amplification
1. Primer design
According to the gamma-interferon whole gene sequence with UNIPORT accession number P01579 and codon optimization of the gene sequence aiming at escherichia coli, a specific primer for amplifying a gamma-interferon IA3 antigen gene DNA fragment is designed according to the optimized DNA sequence, double enzyme cutting sites of BamHI and XbaI are introduced into the tail ends of an upstream primer and a downstream primer, and the length of an expected amplification fragment is about 162bp. Specific primers designed to amplify the a antigen gene were as follows:
an upstream primer IA3-F:5 'cggggatccaaaaagaaacgcgatttgatttgaa-3' (shown as SEQ ID NO: 9);
downstream primer IA3-R:5' of-;
and (3) PCR amplification: and (3) amplifying by using a PCR method by using DNA as a template and a designed specific primer to obtain an IA3 antigen PCR amplification product.
The PCR reaction system is as follows: 20 μ L system: mg (magnesium) 2+ 1.5mmol/L, dNTP 0.2mmol/L, taq enzyme 1U, primers each 5pmol/L, sample DNA 100-200 ng, and the balance of ultrapure water.
The PCR reaction program is: 10min at 95 ℃; 10 cycles of 94 ℃ 20s,63 ℃ 30s and 72 ℃ lmin; performing 25 cycles at 94 ℃ for 20s,60 ℃ for 30s and 72 ℃ for 1 min; extending for 5min at 72 ℃; storing at 4 deg.C.
2. Detection of PCR products
And (3) identifying the amplified fragment by electrophoresis, detecting the amplified product on 1% agarose gel, wherein the voltage is 10v/cm, and the electrophoresis is performed for about 20 min. The length of the target fragment was marked using DNA molecular marker DL2000 (TAKARA, dalianbao Biopsies), and the result is shown in FIG. 1, in which IA3 amplified fragment is 162bp, which is in accordance with the expectation.
3. Recovery and purification of PCR-specific fragments
Use of
The amplification product was recovered and purified by Gel Extraction Kit (Omega Co.).
3. Construction of recombinant expression vectors
1. Construction of recombinant plasmid pET32a-IA3
Purifying and recovering the correctly identified PCR specific fragment according to the DNA gel recovery kit specification, and carrying out enzyme digestion identification on the recombinant plasmid: extracting plasmid according to the operation steps of a plasmid extraction kit (OMEGA), and performing double enzyme digestion identification by using BamH I and Xba I, wherein the enzyme digestion system comprises the following steps: 10 XBuffer 1. Mu.l, bamHI 0.5. Mu.l, xbaI 0.5. Mu.l, recombinant plasmid 8. Mu.l. The enzyme was cleaved at 37 ℃ for 1.5h.
2. Construction of recombinant expression vector pET32a-IA3
And carrying out double enzyme digestion on the PCR specific fragment and the expression vector pET32a by BamH I and Xba I, and connecting to prepare the recombinant expression vector pET32a-IA3.
Connecting a reaction system: 5.5 ul of the target gene fragment, 2 ul of the expression vector, and 2 XT 4DNA Ligase Mix7.5 ul. Flash separated, mixed well and ligated overnight at 16 ℃. Obtaining a ligation product-recombinant plasmid pET32a-IA3
Transferring 5 mu L of recombinant plasmid pET32a-IA3 into DH5 alpha competent cells, slowly blowing and uniformly mixing, and placing on ice for 30min; thermally shocking at 42 deg.C for 90s, ice-cooling for 2min, adding 445 μ L LB liquid culture medium, shake-culturing at 37 deg.C for 1h at 200-250 r/min, centrifuging at 4000r/min for 2min, and mixing; uniformly coating the bacterial liquid on an LB agar plate containing antibiotics, and placing the bacterial liquid in a thermostat at 37 ℃ after the bacterial liquid is absorbed for overnight culture until a single bacterial colony appears; selecting bacteria, and culturing overnight at 37 ℃ under shaking at 300 r/min.
PCR screening positive clone of bacterial liquid: and (3) carrying out PCR amplification on the IA3 gene by using turbid bacterial liquid as a template. The product was checked for amplification by electrophoresis on a 1% agarose gel.
And (3) identification result: the IA3 gene is cloned to a pET32a vector, expected bands are obtained after PCR and enzyme digestion identification, the length of A in the recombinant plasmid pET32a-IA3 is 162bp, and the sequencing result of pET32a-IA3 is consistent with the optimized sequence of gamma-interferon IA2, which indicates that the construction of the recombinant expression vector is successful.
4. Transformation of
The recombinant expression vector pET32a-IA3 is transformed into E.coli.BL21 expression host bacteria, and positive expression bacteria are screened.
5. Inducible expression
Inoculating the screened positive expression strain containing the recombinant expression vector pET32a-IA3 into an LB culture medium, culturing overnight at 37 ℃, adding IPTG (isopropyl-beta-D-thiogalactoside) into the bacterial liquid to a final concentration of 0.4mmol/L when the bacterial liquid OD600nm =0.6 is subjected to amplification culture according to a ratio of 1.
6. Purification of
Centrifuging the bacteria solution after induced expression for 10min at 12000r/min, enriching thalli, resuspending the thalli by using 20mmol/L Tris-HCl buffer solution with the pH value of 8.0 according to the volume ratio of 1.
Example 4 antigen immunoreactivity identification
1. Identification of immunological Activity
1. The recombinant human gamma-interferon antigens IA1, IA2 and IA3 prepared by the method are detected by adopting a human gamma-interferon detection kit of a Wantai organism, and the sample concentration is 1ng/ml. The detection result is positive, and the OD value is greater than the positive quality control. It is demonstrated that the recombinant human interferon-gamma antigen IA1 obtainable by the method of the present invention has immunological activity. The results are shown in Table 1 below:
TABLE 1 immunological Activity of gamma-interferon antigens IA1, IA2, IA3
2. The result of immunoblotting identification (WB) using the labeled antibody of the human interferon-gamma detection kit of a Wantai organism is shown in FIG. 4, which shows that the recombinant human interferon-gamma antigen IA1 has immunological activity and can be used for animal immunization.
2. Stability of
The recombinant human interferon gamma-interferon for injection is subjected to freeze-thaw stability detection by adopting an atlas interferon gamma-detection kit, the recombinant injection antigen and the recombinant IA1 protein are subjected to gradient dilution for activity detection after purification, re-melting at-20 ℃ after 7 days, placing at-20 ℃ and 4 ℃ for 14 days after 14 days, and are subjected to A2000 on-machine test after being respectively diluted by 10000, 20000, 40000, 80000 and 160000 times, and the results are shown in Table 2.
The human gamma-interferon for recombinant injection has poor freeze-thaw stability, and the activity is lost by 80% after freeze-thawing for 2 times; the average reduction amplitude of the recombinant protein IA1 is within 9 percent, and the freeze-thaw stability of the recombinant protein IA1 is superior to the full length of the gamma-interferon for injection in a controllable range.
TABLE 2 recombinant protein IA1 stability
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. The gamma-interferon recombinant protein is characterized in that the amino acid sequence is shown as SEQ ID NO: shown at 11.
2. A nucleic acid encoding the recombinant gamma-interferon protein of claim 1.
3. The nucleic acid of claim 2, having the sequence as set forth in SEQ ID NO: 2.
4. A recombinant vector comprising a vector backbone and the nucleic acid of claim 2.
5. A host, characterized in that it comprises any one or more of the following I) to II):
i) Chromosomally integrating the nucleic acid of claim 2 or 3;
II), transfection or transformation of the recombinant vector according to claim 4.
6. The host of claim 5, wherein the host is E.
7. The method for producing a recombinant protein of gamma-interferon according to claim 1, which comprises culturing the host of claim 5 or 6 to obtain a culture containing the recombinant protein of gamma-interferon.
8. The application of any one of the following i) to v) in preparing a gamma-interferon antibody or a gamma-interferon detection product:
i) The recombinant gamma-interferon protein according to claim 1;
ii) the nucleic acid of claim 2 or 3;
iii) The recombinant vector of claim 4;
iv) the host of claim 5 or 6;
v) a culture containing the recombinant protein of gamma-interferon produced by the production method according to claim 7.
9. A interferon-gamma antibody produced using the interferon-gamma recombinant protein according to claim 1 as an antigen.
10. A γ -interferon assay product, starting from which comprises the γ -interferon antibody of claim 9.
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