CN112521461B - Preparation of hepatitis A virus recombinant protein and rapid detection method thereof - Google Patents
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Abstract
The invention relates to the field of biotechnology, and discloses a preparation method and a rapid detection method of hepatitis A virus recombinant protein. The invention selects two dominant epitopes of hepatitis A virus antigen, uses a flexible segment (four glycines) as a connecting short peptide sequence, and repeatedly connects the two dominant epitopes in series to form a recombinant protein. And adopting CHO cell preferred codon to convert the recombinant antigen amino acid sequence into corresponding nucleotide sequence, chemically synthesizing the nucleotide sequence and constructing a recombinant expression vector, thereby improving the expression quantity of the recombinant antigen in CHO cells. In addition, a colloidal gold diagnosis platform with high specificity and high sensitivity is established by adopting and based on the recombinant protein.
Description
The technical field is as follows: the invention relates to the field of biotechnology, in particular to a hepatitis A virus recombinant antigen, a preparation method thereof, a preparation method of a polyclonal antibody of the recombinant antigen, and a rapid diagnosis platform for establishing the hepatitis A virus by using the recombinant antigen.
Background art: viral hepatitis A, abbreviated as hepatitis A and hepatitis A, is an infectious disease caused by Hepatitis A Virus (HAV) and mainly caused by liver inflammation. Hepatitis A is a common infectious disease in clinic, generally, hepatitis A is only a common hepatitis, and in a few cases, severe hepatitis develops. Therefore, accurate diagnosis can be carried out in the early stage of hepatitis A, and the hepatitis A can be treated correspondingly in time, so as to prevent further severe hepatitis. Therefore, early and rapid diagnosis is the key and key point for preventing and treating the disease.
Currently, commonly used methods for diagnosing viral hepatitis a include enzyme-linked immunosorbent assay (ELISA), colloidal gold method established with natural viruses, polymerase chain reaction technology, and the like. The enzyme-linked immunosorbent assay (ELISA) detects the antigen or the antibody by enzyme-labeled color development by using the principle of specific combination of the antigen and the antibody. Although IgM antibodies against hepatitis A virus can be detected by ELISA method, it is possible to judge whether or not hepatitis A virus is infected. However, enzyme-linked immunosorbent assay (ELISA) is time-consuming and tedious in operation, which relatively limits the large-scale production and release. And the raw materials used by the traditional enzyme-linked immunosorbent assay (ELISA) are natural antigens or vaccines of hepatitis A virus, which causes low specificity in detection. Similarly, the colloidal gold detection methods commonly found in the market are all established by taking natural viruses or vaccines as raw materials, and have the phenomenon of low specificity, which easily causes the misjudgment of diseases and receives unnecessary treatment means. The polymerase chain reaction technology is a means for detecting by a means of amplifying specific genes, a section of highly conserved sequence of the hepatitis A virus is selected by the method, and although the polymerase chain reaction technology has the characteristics of high sensitivity and strong specificity, the operation is complex and time-consuming.
Therefore, the establishment of a method for diagnosing the viral hepatitis A infection, which is rapid, simple and convenient to operate and high in specificity, has great significance.
The invention content is as follows:
one of the objects of the present invention is to provide a DNA sequence of the hepatitis A virus recombinant protein AV 56.
The invention also aims to provide a hepatitis A virus recombinant protein AV56 efficiently expressed by CHO cells, and provides a specific antigen for the subsequent establishment of a rapid detection method of the hepatitis A virus.
It is another object of the present invention to provide a polyclonal antibody that specifically recognizes the recombinant protein.
The fourth purpose of the invention is to provide a method for diagnosing the viral hepatitis A infection, which has the advantages of rapidness, simple operation and high specificity.
The invention is realized by the following experimental scheme:
1) comparing the gene sequence of the hepatitis A virus with the characteristic epitopes of other homologous pathogenic microorganisms, simulating the dominant epitope of the hepatitis A virus by a computer, finally selecting two dominant antigen epitope sequences of the hepatitis A virus to be connected by a flexible segment (four glycines), taking the sequence obtained after repeated series connection as a target sequence, and synthesizing a corresponding nucleotide sequence by adopting CHO cell preferred codons.
2) And directionally inserting the synthesized nucleotide sequence into a eukaryotic expression vector to construct a recombinant eukaryotic expression vector.
3) Transforming the recombinant eukaryotic expression vector obtained in the step 2) into escherichia coli DH5 alpha competent cells, and obtaining a recombinant expression plasmid through screening and identification.
4) Transient transformation of the recombinant expression plasmid obtained in the step 3) to CHO cells was performed, and cell supernatants were collected 7 days later.
5) Purifying the cell supernatant obtained in the step 4) to obtain the recombinant protein AV 56.
6) Immunizing goats with the AV56 recombinant protein purified in the step 5), preparing polyclonal antibodies, and purifying the prepared polyclonal antibodies.
7) Labeling the AV56 recombinant protein purified in the step 5) with colloidal gold particles.
8) Coating the polyclonal antibody purified in the step 6) as a C line.
9) Mouse anti-human IgM monoclonal antibodies (Hangzhou xian till Biotech Co., Ltd.) were coated as T-lines, respectively.
10) And assembling the colloidal gold test strip, and detecting the serum to be detected by using the colloidal gold test strip. The clinical use of the colloidal gold is determined according to the color change of the colloidal gold.
The plasmid vector is a vector tool which is most widely applied in the field of molecular biology, and CHO cells are important expression production systems in the field of biology. The method is convenient to operate, has no potential safety hazard to the environment and operators, expresses the obtained fusion antigen, has no pathogenicity and infectivity, and repeats the two dominant antigen epitopes for multiple times, thereby not only improving the immunogenicity in the preparation process of the polyclonal antibody, but also increasing the labeling effect of the colloidal gold. Meanwhile, the detection of the acute infection stage of the hepatitis A virus can be realized by coating the mouse anti-human IgM monoclonal antibody, so that the hepatitis A virus can be conveniently and timely treated correspondingly, and further severe hepatitis can be prevented from being developed.
In the following examples, although the design idea of the present invention is described in more detail, these descriptions are only simple descriptions of the design idea of the present invention, but not limitations of the design idea of the present invention, and any combination, addition, or modification without departing from the design idea of the present invention falls within the scope of the present invention.
Example 1: hepatitis A virus dominant antigen epitope sequence selection
The hepatitis A virus is taken as a target antigen, the biological software DNAssist2.0 is utilized to analyze the hydrophilicity and antigenicity of an antigen epitope sequence, the specificity and the sensitivity are considered, and two dominant antigen epitope sequences are finally selected, wherein the dominant antigen epitope sequences are specific sequences of hepatitis A virus protein and have no obvious homology with other protein sequences.
Example 2: hepatitis A virus dominant antigen epitope sequence series connection and optimization
In order to improve the labeling effect of the colloidal gold and the requirement of the preparation titer of the protein polyclonal antibody, two sections of hepatitis A virus dominant antigen epitopes are connected through a section of flexible segment, the connecting sequence is repeated for 3 times through a section of flexible segment, and in order to improve the expression quantity of the recombinant protein in CHO cells, a CHO cell preferred codon is adopted to synthesize a corresponding nucleotide sequence to obtain the amino acid sequence of the recombinant protein, wherein the specific sequence is shown as a sequence table SEQ ID No. 1. The connected recombinant protein amino acid sequence is converted into a corresponding nucleotide sequence, the specific coding sequence is shown as a sequence table SEQ ID No. 2, nucleotide sequences corresponding to enzyme cutting sites BamHI and EcoRI are respectively added at the upstream and downstream of the sequence, and the sequence is synthesized by Nanjing Kingsry Biotech Co. The synthesized target gene is cloned in pMD19-T vector (Takara Bio-engineering Co., Ltd.).
Example 3: construction of recombinant protein expression vectors
The pMD19-T vector and pTT5 vector containing the target gene were digested separately with restriction enzymes BamHI and EcoRI (Bao bioengineering Dalian Co., Ltd.) at 37 ℃ for 12 hours, and the digested fragments were enriched by 1% agarose gel electrophoresis and then gel recovered (the target gene recovery and the digestion products were recovered using Ningbo Zhongding Biotechnology Co., Ltd. gel recovery kit). The recovered target gene fragment and pTT5 vector fragment were ligated at 4 ℃ for 12 hours using T4 ligase (Takara Shuzo Co., Ltd.) in a ratio, and the ligation product was transformed into DH 5. alpha. competent cells, plated on LB plates containing ampicillin resistance (50. mu.g/mL), and cultured overnight at 37 ℃.
The monoclonal strains picked on the plate are inoculated into LB liquid culture medium containing ampicillin resistance (50 mu g/mL), after shaking culture at the constant temperature of 37 ℃ for 5 hours, strain plasmids are extracted (by using plasmid purification kit of Ningbo Zhongding biotechnology Limited company), and the correct recombinant expression vector pTT5-AV56 is obtained after BamHI and EcoRI double enzyme digestion identification.
Preparation of DH5 α competent cells: adding 100 μ l of Escherichia coli DH5 α bacterial liquid into LB culture medium (2mL), shake culturing at 37 deg.C for 3-4 hr until OD600 is 0.6, adding 100 μ l of activated bacterial liquid into LB culture medium (6mL), shake culturing at 37 deg.C for 1.5 hr until OD600 is 0.6, collecting 1mL bacterial liquid, centrifuging at low temperature, removing supernatant, adding 160 μ l CaCl-containing precipitate2Blowing glycerol solution, homogenizing at 5000rpm, centrifuging at 4 deg.C for 3min, discarding supernatant, adding 160 μ l CaCl2Glycerol solution, and homogenizing by blowing to obtain DH5 alpha competent cells.
CaCl2-glycerol solution formulation: CaCl2Final concentration 1mol/L, glycerol final volume 10%
Example 4: transfection and purification of eukaryotic animal cells by recombinant expression vector
The constructed recombinant expression vector pTT5-AV56 was transfected into CHO-K1 cells. CHO-K1 cells were plated at 1X 10 the day before transfection6Density passaging/ml to ensure cell viability at transfection day 2X 106Transfection was performed per ml.3.2 mu g of recombinant expression vector is added into each ml of the transfection system, and 4.8 mu g of transfection reagent PEI (Polyscience) is added into each ml of the transfection system, and the mixture is shaken up while adding. After culturing at 37 ℃ and 120rpm of a 6% carbon dioxide shaker for 4 hours, 1% 500mM VPA (sigma) and 1% 30g/L L-cysteine hydrochloride (Solebao Biotech Co., Ltd.) were added, and after culturing at 32 ℃ and 120rpm of a 6% carbon dioxide shaker for 6 days, the supernatant was centrifuged and collected, passed through a nickel agarose affinity column (Hezhou Tiandi and Biotech Co., Ltd.), a 20mM imidazole solution was used for removing foreign proteins, a 300mM imidazole solution was used for eluting the target protein, and after collecting the solution, the solution was allowed to stand at 4 ℃ for 30 minutes, transferred into a dialysis bag having a cut-off molecular weight of 10kD to 12kD, and dialyzed overnight in PBS (10mmol/L, pH 7.4). Immediately taking out after dialysis and subpackaging, and storing at-20 ℃ for later use.
20mM imidazole preparation: imidazole 1.36g, add 10mmol/L, pH7.4 PBS solution to dissolve to 1000 mL.
300mM imidazole preparation: imidazole 10.2g, add 10mmol/L, pH7.4 PBS solution to dissolve to make volume 500 mL.
Example 5: ELISA detection
Diluting the purified AV56 with coating solution to a final concentration of 1 μ g/mL, adding 100 μ L/well into an ELISA plate (Stannless department of bioengineering, Ltd.), coating overnight at 4 deg.C, washing with washing solution by DEM-3 type plate washing machine (Daan Gene, Inc. of Zhongshan university) for 1 time, adding 200 μ L of blocking solution after plate washing, incubating at 37 deg.C for 1 hour, and washing with washing solution for 1 time. Then adding clinically confirmed hepatitis A negative serum and hepatitis A IgM positive serum respectively, incubating at 37 ℃ for 35 minutes, washing with a washing solution for 3 times, adding HRP-labeled rabbit anti-human IgM antibody (Hangzhouxian till Biotechnology Co., Ltd.), incubating at 37 ℃ for 35 minutes, and washing with a washing solution for 4 times. And adding the solution A and the solution B, then adding the stop solution, carrying out OD value measurement after the blank control hole is zero, and the result shows that the OD value of the IgM positive serum hole is 2.8 times that of the negative serum hole.
Coating liquid: na (Na)2CO3 1.59g，NaHCO32.93g, and then ultrapure water was added thereto to make the volume 1000mL (pH 9.6).
Sealing liquid: 3g of illite skim milk powder is dissolved by adding 10mmol/L of PBS solution with pH value of 7.4 to be 100 mL.
Washing liquid: na (Na)2HPO4.12H2O 2.68g,NaH2PO4.2H20.39g of O, 8.5g of NaCl, and 200.5 mL of Tween, and adding ultrapure water to the volume of 1000mL (pH7.4).
The color developing solution A is prepared by dissolving 200mg of TMB in 100mL of absolute ethyl alcohol and adding ultrapure water to reach the constant volume of 1000 mL.
Color developing solution B containing citric acid 2.1g and Na2HPO4.12H2O71 g, and adding ultrapure water to the solution to make the volume reach 1000 mL.
When in use: 1mL of developing solution A +1mL of developing solution B + 0.4. mu.L of 30% H2O2
Stopping liquid: 2M H2SO421.7mL of concentrated H2SO4Adding ultrapure water to the solution until the volume is 1000 mL.
Example 6: preparation of recombinant protein AV56 sheep polyclonal antibody
A first basal immunization was performed by taking 300. mu.g of AV56 recombinant protein, emulsifying with Freund's complete adjuvant (total 1ml), and injecting into one sheep at multiple points intradermally. After 20 days, a second immunization (booster) was performed, i.e., 150. mu.g of AV56 recombinant protein was emulsified with Freund's incomplete adjuvant (total 1ml) and injected into the skin at multiple points. Then, the immunization is performed once every 15 days in the same way as the second immunization, 5ml of blood is collected from the ear artery 10 days after the 4 th boosting immunization, and the AV56 polyclonal antibody is obtained through purification and the titer is detected through ELISA. The specific procedure was the same as in example 6, except that AV56 polyclonal antibody was added as a primary antibody, similarly to the IgM positive serum for viral hepatitis A in example 6. Carotid blood was collected 10 days after the fifth booster immunization.
Example 7: purification of recombinant protein AV56 rabbit polyclonal antibody
The agarose affinity medium Protein G column (Nanjing King Shirui Biotech Co., Ltd.) was equilibrated to room temperature, preheated for 20 minutes by a computer nucleic acid Protein detector (Shanghai Huxi analytical Instrument Co., Ltd.), and washed with 10mmol/L of PBS solution having pH of 7.4 until the absorbance A of the computer nucleic acid Protein detector showed 0. After centrifugation at 12000rpm for 5 minutes, the supernatant was applied to a 0.45 μm filter, and then 10mmol/L PBS (pH7.4) was added thereto, and the mixture was subjected to column washing until the absorbance A in a computer nucleic acid protein detector showed 0. Elution was performed with 0.1mol/L glycine solution at pH 3.0. And collecting the eluate, and adding 0.5mol/L Tris-HCl buffer solution with the pH value of 8.5 to neutralize the eluate to the pH value of 7.0, so as to obtain the recombinant protein AV56 goat polyclonal antibody.
Preparing a glycine solution: 7.5g of glycine is dissolved in ultrapure water, the volume is adjusted to 800ml, and concentrated HCl 6-8 is added to adjust the pH value to 3.0 Tris-HCl buffer solution to prepare: 75.4g of Tris was dissolved in ultrapure water under stirring, and the volume was adjusted to 1000ml, and about 10ml of concentrated HCl was added to adjust the pH to 8.5.
Example 8: recombinant protein AV56 labeled colloidal gold particles
Adding 0.2mol/L potassium carbonate solution 10 μ L into 5ml 0.01% colloidal gold solution, mixing well, adding 100 μ g hepatitis A virus recombinant protein AV56, mixing well, standing at room temperature for 2 hr, adding 500 μ L10% BSA (bovine serum albumin) solution, sealing, standing at room temperature for 1 hr, centrifuging (10000rpm, 20min), discarding supernatant, and dissolving precipitate with 500 μ L redissolution. The dissolved gold solution was uniformly sprayed on a 6mm wide glass fiber by a gold spraying and film-drawing instrument (Shanghai gold-labeled Biotech Co., Ltd.) and then dried by blowing at 37 ℃ for 1 hour in an electric hot air drying oven (Shanghai-Hengscientific Instrument Co., Ltd.).
The relevant solution formulation is as follows:
0.01% colloidal gold solution: 1ml of 1% chloroauric acid solution, 1.4ml of 1% citric acid solution, adding ultrapure water, heating, dissolving, reacting and fixing the volume to 100 ml.
1% chloroauric acid solution: AuCL3.HCl.4H21g of O powder is dissolved by adding ultrapure water and the volume is adjusted to 100 ml.
1% citric acid solution: 1g of citric acid crystal is dissolved by adding ultrapure water and the volume is adjusted to 100 ml.
Compounding the solution: 6.057g of Tris base is dissolved in 800ml of ultrapure water, the pH is adjusted to 8.0 by using a proper amount of HCL, and the volume is adjusted to 1000ml by adding the ultrapure water.
Example 9: preparation of test paper for diagnosing virus hepatitis A
The hepatitis A virus recombinant protein AV56 polyclonal antibody is diluted by coating liquid to a final concentration of 1mg/ml, and is uniformly coated on a nitrocellulose membrane (Sartorius, CN140) by a gold spraying and membrane scribing instrument (Shanghai gold-labeled Biotech Co., Ltd.) according to 1. mu.l/cm to obtain a C line.
The mouse anti-human IgM monoclonal antibody (Hangzhou xian till Biotechnology Co., Ltd.) was diluted with a coating solution to a final concentration of 1mg/ml, and was uniformly coated on a nitrocellulose membrane (Sartorius, CN140) as a T line by a gold spraying and membrane scribing instrument (Shanghai gold-labeled Biotechnology Co., Ltd.) in an amount of 1. mu.l/cm.
After the completion of the film-scribing, the nitrocellulose membrane was air-dried at 37 ℃ for 30 minutes in an electric hot air drying oven (Shanghai-Hengyu scientific instruments Co., Ltd.).
The nitrocellulose membrane, the colloidal gold pad, the sample pad and the absorbent paper are assembled on a PVC pad in sequence, then cut into strips with the width of 4mm, and then packed into a card shell and compressed.
Coating liquid: na (Na)2HPO4.12H2O17.9 g, and double distilled water is added to the solution to reach a volume of 1000mL (pH 8.0).
Example 10: operation steps and standards of test paper strip for diagnosing virus A hepatitis
10 parts of clinical IgM positive serum and 30 parts of clinical negative IgM serum of a hepatitis A patient are taken, diluted 1000 times by using normal saline, loaded at 100 mu L/well, placed at room temperature for 10min, read the color depth values of two lines of C and T by using a colloidal gold reader (Hangzhou Hunzan technology Co., Ltd.), and calculate T/(T + C). The reading results are shown in the attached table I. As shown by experimental results, the colloidal gold test strip established on the basis of the hepatitis A virus recombinant antigen AV56 prepared by the invention can quickly and effectively identify and diagnose patients and normal people with the hepatitis A virus.
Example 11: specificity test of test paper
According to the operation method of the hepatitis A virus test strip, the test strip is used for detecting 100 negative serums (determined clinically), false positive does not appear, and the coincidence rate is 100%.
According to the operation method of the hepatitis A virus test strip, the test strip is used for detecting 10 parts of hepatitis B virus positive serum specimen, 10 parts of hepatitis C virus positive serum specimen, 10 parts of AIDS virus positive serum and the like, and 10 parts of helicobacter pylori virus positive serum, wherein the positive production does not exist.
The result shows that the colloidal gold rapid detection test strip prepared by the hepatitis A virus recombinant antigen AV56 does not cause cross reaction with common basic diseases and other viral diseases of human and has good specificity.
Attached watch 1
1, SEQ ID No: hepatitis A virus recombinant protein amino acid sequence
2, SEQ ID No: hepatitis A virus recombinant protein nucleotide sequence
Sequence listing
<110> Hangzhou xian Zhi Biotechnology Co., Ltd
<120> preparation of hepatitis A virus recombinant protein and rapid detection method thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 101
<212> PRT
<213> Artificial Synthesis (Artificial Sequence)
<400> 1
Leu Thr Pro Leu Ser Thr Gln Met Met Arg Asn Glu Gly Gly Gly Gly
1 5 10 15
Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr Val His Gly
20 25 30
Gly Gly Gly Leu Thr Pro Leu Ser Thr Gln Met Met Arg Asn Glu Gly
35 40 45
Gly Gly Gly Ser Glu Gly Pro Leu Lys Met Glu Glu Lys Ala Thr Tyr
50 55 60
Val His Gly Gly Gly Gly Leu Thr Pro Leu Ser Thr Gln Met Met Arg
65 70 75 80
Asn Glu Gly Gly Gly Gly Ser Glu Gly Pro Leu Lys Met Glu Glu Lys
85 90 95
Ala Thr Tyr Val His
100
<210> 2
<211> 303
<212> DNA
<213> Artificial Synthesis (Artificial Sequence)
<400> 2
ctgaccccgc tgagcaccca gatgatgcgc aacgaaggcg gcggcggcag cgaaggcccg 60
ctgaaaatgg aagaaaaagc cacctatgtg catggcggcg gcggcctgac cccgctgagc 120
acccagatga tgcgcaacga aggcggcggc ggcagcgaag gcccgctgaa aatggaagaa 180
aaagccacct atgtgcatgg cggcggcggc ctgaccccgc tgagcaccca gatgatgcgc 240
aacgaaggcg gcggcggcag cgaaggcccg ctgaaaatgg aagaaaaagc cacctatgtg 300
cat 303
Claims (5)
1. A hepatitis A virus recombinant antigen has an amino acid sequence shown in SEQ ID No. 1.
2. A nucleotide capable of coding the hepatitis A virus recombinant antigen of claim 1, the sequence of which is shown in SEQ ID No. 2.
3. A plasmid vector comprising the nucleotide of claim 2.
4. A strain comprising the plasmid vector of claim 3.
5. Use of the recombinant hepatitis a virus antigen of claim 1 in the preparation of a test strip for the diagnosis of hepatitis a.
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| CN101875942A (en) * | 2009-04-30 | 2010-11-03 | 上海泽润生物科技有限公司 | Hepatitis A virus genome complete sequence |
| CN105198969A (en) * | 2015-11-02 | 2015-12-30 | 四川农业大学 | B-cell epitope of VP(viral protein)3 of DHAV (duck hepatitis A virus)-1 as well as identification method and application of B-cell epitope |
| CN106632619A (en) * | 2016-12-02 | 2017-05-10 | 杭州贤至生物科技有限公司 | Influenza A virus recombinant protein and preparation of monoclonal antibody thereof |
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