CN115838420B

CN115838420B - Preparation method of soluble HCV recombinant protein and antibody detection reagent prepared by preparation method

Info

Publication number: CN115838420B
Application number: CN202211344465.XA
Authority: CN
Inventors: 詹先发; 柳静
Original assignee: Beijing Key Biotechnology Co ltd
Current assignee: Beijing Key Biotechnology Co ltd
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2023-05-05
Anticipated expiration: 2042-10-31
Also published as: CN115838420A

Abstract

The invention provides a preparation method of soluble HCV recombinant protein and an antibody detection reagent prepared by the same, wherein the HCV recombinant protein comprises truncated NS3 protein and truncated Core protein, can improve the immune response degree of an organism to HCV antigen, and is beneficial to development of anti-KCV vaccine and screening of HCV diagnostic antibodies; optimizing the preparation process of the HCV recombinant protein, comprising designing the nucleotide sequence of the recombinant protein by using the preferential codons of escherichia coli, and improving the production capacity; optimizing the process steps and operating parameters in the preparation process, and providing a fermentation medium with good production performance, thereby obtaining high-purity recombinant protein; based on the HCV recombinant protein, the immune mice obtain monoclonal antibodies with high affinity to target antigens, and can effectively recognize HCV; by using the antibody, a detection kit is constructed, and the HCV infection condition can be effectively detected, so that a detection means with high specificity, high sensitivity and good stability is provided.

Description

Preparation method of soluble HCV recombinant protein and antibody detection reagent prepared by preparation method

Technical field:

the invention belongs to the field of disease diagnosis, and particularly provides a preparation method of soluble HCV recombinant protein and an antibody detection reagent prepared by the same.

The background technology is as follows:

chronic hepatitis c virus (Hepatitis C virus, HCV) infection is one of the leading causes of liver fibrosis, cirrhosis, and hepatocellular carcinoma (hepatocellular carcinoma, HCC). Although effective treatment can be performed using direct-acting antiviral Drugs (DAA), DAA can satisfactorily treat 95% or more of HCV-infected persons, thereby reducing the risk of death from liver cancer and cirrhosis, about 7100 tens of thousands of persons worldwide still have long-term infection of the virus, thus facing the risk of developing end-stage liver disease (World Health organization report. World Health Organization; geneva, switzerland: 2017). DAA drugs are too expensive and thus large scale treatment presents socioeconomic barriers, especially in low and medium income countries, and are therefore only suitable for a small percentage of infected patients in developing and developed countries. In addition, DAA treatment does not prevent or arrest the occurrence of cancerous lesions, and HCC recurrence rate may even increase after DAA treatment (Sanduzzi-Zamparelli M., boix L., leal C., reig M. Hepatocellar carcinoma recurrence in HCV patients treated with direct antiviral agents. Viruses.2019; 11:406). In addition, reinfection still exists in high-risk groups, so that satisfactory effects are difficult to obtain for treating hepatitis C by means of medicines alone. Thus, the lack of diagnosis of HCV remains a serious challenge, and the development of effective novel diagnostic agents for HCV is particularly important.

Conventional diagnostic systems for HCV infection involve serological biomarkers and molecular detection. The serological biomarkers may detect anti-HCV antibodies or HCV antigens, while molecular detection may detect HCV RNA. Currently, the use of serological assays to detect anti-HCV antibodies is the first step in HCV detection algorithms, as they are easier to handle and more economical than molecular assays.

The gold standard serological test for detection of anti-HCV antibodies is a third generation Enzyme Immunoassay (EIA), namely the ORTHO HCV 3.0 version of ELISA test system (Ortho Diagnostic Systems, usa) and the Murex anti-HCV 4.0 (Murex Diagnostics, uk) multiplex assay for anti-HCV antibodies using different core, NS3, NS4 and NS5 recombinant antigens. Variants of EIA include chemiluminescent immunoassays (CLIA), as is the case with the Foresight HCV EIA test (ACONLabs, US), enzymatic CLIA, such as luminol-H ₂ O ₂ Horseradish peroxidase (HRP) and Chemiluminescent Microparticle Immunoassay (CMIA). Other relevant HCV antibody detection techniques, including fluorescent immunoassays, anti-HCV antibody detection protein chip methods, etc., have also been used clinically.

Since HCV was found, researchers have conducted intensive studies on the genome, proteome and metabonomics of HCV, and found that HCV genome is about 9.6kb long, encodes a multimeric protein precursor of about 3000 amino acid residues long, and generates 4 structural proteins (C, E1, E2, P7) and 6 non-structural proteins (NS 2, NS3, NS4A, NS B, NS5A, NS B) under the action of host signal peptidase and virus self-encoded protease, wherein Core, NS3 antigens have good complementarity in antigen reactivity, and the combined detection rate average can be about 99%, which is an important segment for research of HCV diagnostic reagents, so that researchers have proposed the design and preparation of Core-NS3 fusion proteins for HCV detection (CN 113640519 a). In addition, CN105254724a proposes the construction of truncated HCV ns3 antigen of hepatitis c virus for HCV detection; CN102072957a proposes that the specific HCV recombinant multi-antigen component (i.e. gene fragment containing Core, NS3, NS4, NS 5) is used to label magnetic particles by using microparticle chemiluminescence immunoassay technology, and the specific anti-human IgG is used to label isoluminol, and the reaction system adopts a two-step method to detect HCV.

Although the use of HCV Core and NS3 antigens to construct fusion proteins or related antibodies to detect HCV viruses has been disclosed in the prior art, existing methods either use full-length proteins, with ambiguous epitopes; or targeting a single type of antigen, it is difficult to effectively improve the detection accuracy, thereby limiting its application in hepatitis c diagnosis. Thus, the present application provides a method for preparing a soluble HCV recombinant protein, in which a fusion protein comprising HCV truncated Core and truncated NS3 antigen is constructed, and monoclonal antibodies are screened using the fusion protein, for preparing a hepatitis c diagnostic kit, which can improve the sensitivity and specificity of detection.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a soluble HCV recombinant protein, wherein the amino acid sequence of the HCV recombinant protein is shown as SEQ ID NO.1, and the preparation method comprises the following steps: constructing an expression vector carrying the HCV recombinant protein gene; causing the expression vector to be in an E.coli expression system; culturing Escherichia coli by high-density fermentation; and (3) collecting escherichia coli thalli, and separating and purifying to obtain the soluble HCV recombinant protein.

The soluble HCV recombinant protein provided by the invention is a fusion protein, which comprises truncated NS3 protein and truncated Core protein, and the two proteins have good complementarity in antigen reaction, and are key sections of HCV diagnostic reagents, so that the use of the fusion protein comprising both NS3 protein and Core protein sections can improve the detection accuracy and sensitivity, and lay a good foundation for the subsequent screening of high-specificity antibodies; the fusion protein can be used for immunizing mice to obtain detection antibodies, can be used for potential HCV vaccines, can excite effective immune response in organisms, and provides a basis for developing novel HCV preventive medicines. In addition, the fusion protein is constructed by using the truncated protein fragment, so that the operation difficulty of molecular biology can be reduced, the absorption efficiency and bioavailability in the organism can be improved, the immune function of experimental animals can be mobilized, and the guarantee is provided for screening and obtaining high-specificity antibodies.

Furthermore, the nucleotide sequence of the HCV recombinant protein is shown as SEQ ID NO. 2. The nucleotide sequence is designed according to the preferential codons of the escherichia coli, so that the expression efficiency in an escherichia coli expression system can be improved, and the preparation and purification processes of recombinant proteins are facilitated.

Further, the preparation method further comprises the following steps: amplifying the recombinant protein gene by PCR, introducing Xho I and EcoR I enzyme cutting sites at two ends of a nucleotide sequence of the recombinant protein gene, connecting the recombinant protein gene to a pET28a vector after enzyme cutting, and electrically transforming the recombinant protein gene into DH5 alpha competent cells; screening positive clones, and performing enzyme digestion and sequencing to identify correct clones; connecting the recombinant protein gene to a prokaryotic expression vector pGEX 6P-1, introducing the prokaryotic expression vector into escherichia coli BL21, performing amplification culture, inoculating to a bioreactor, adding a fermentation medium, culturing at 37 ℃, and stirring at a speed of 350-450r/min, wherein the ventilation rate is 0.8-1.5L/L.min; adding IPTG to induce for 16 hours, and centrifugally collecting thalli; the thallus is crushed by ultrasonic, protein A is used for purifying Protein, and the soluble HCV recombinant Protein is obtained.

Further, the fermentation medium comprises 10g/L of tryptone, 5g/L of yeast extract, 2.34g/L of monopotassium phosphate, 3.58g/L of monopotassium phosphate, 10g/L of sodium chloride, 0.64g/L of magnesium sulfate heptahydrate, 0.56g/L of ferric chloride hexahydrate, 10g/L of glycerol and 5g/L of glucose.

A monoclonal antibody specifically recognizing HCV is provided, wherein the heavy chain variable region of the antibody comprises CDR-H1-3 with the amino acid sequence shown as SEQ ID No. 3-5; the light chain variable region of the antibody comprises CDR-L1-3 with an amino acid sequence shown as SEQ ID No. 6-8.

Further, the heavy chain variable region of the antibody has an amino acid sequence shown in SEQ ID No. 9.

Further, the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID No. 10.

Further, the preparation method of the antibody comprises the following steps: immunizing a mouse with the HCV recombinant protein according to any one of claims 1 to 3, and collecting, purifying and screening the mouse ascites.

The monoclonal antibody can recognize HCV with high specificity, improves the sensitivity and specificity of hepatitis C diagnosis, provides diagnosis basis for early detection and treatment of hepatitis C, and further reduces the treatment period and economic burden of patients.

A kit for detecting hepatitis C virus is provided, which is characterized by comprising the monoclonal antibody disclosed by the invention.

Provides the application of the monoclonal antibody in preparing a kit for detecting the hepatitis C virus.

Advantageous effects

The application provides a preparation method of soluble HCV recombinant protein and an antibody detection reagent prepared by the preparation method, which have the following advantages:

(1) The HCV recombinant protein comprises truncated NS3 protein and truncated Core protein, can improve the immune response degree of an organism to HCV antigen, and is beneficial to the development of anti-KCV vaccine and the screening of HCV diagnostic antibodies.

(2) Optimizing the preparation process of the HCV recombinant protein, comprising designing the nucleotide sequence of the recombinant protein by using the preferential codons of escherichia coli, and improving the production capacity; optimizing the process steps and operating parameters in the preparation process and providing a fermentation medium with good productivity, thereby obtaining recombinant proteins with high purity.

(3) Monoclonal antibodies with high affinity to the target antigen are screened and obtained, and HCV can be effectively identified.

(4) The antibody and the related kit can effectively detect HCV infection, and provide a detection means with high specificity, high sensitivity and good stability.

Drawings

Fig. 1: identifying an electrophoresis chart by gene digestion of HCV recombinant proteins;

fig. 2: SDS-PAGE electrophoresis of recombinant proteins;

Detailed Description

The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.

The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.

Example 1 design and preparation of HCV recombinant proteins

1.1 design of HCV recombinant proteins

According to the HCV core and NS3 gene sequences published by NCBI database, truncated amino acid sequences of the core region and NS3 are respectively obtained by screening by reference to published HCV antibody epitopes through sequence comparison, analysis and simulation calculation, and flexible linker (GGGGS) is adopted between the two ₅ And (3) connecting to obtain the HCV recombinant protein, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 1. According to the preference of the escherichia coli codon, the nucleotide sequence of the fusion protein is designed and synthesized as shown in SEQ ID NO. 2, and the gene sequence is obtained by an artificial synthesis method.

1.2 preparation of HCV recombinant proteins

Amplifying a recombinant protein gene sequence by PCR, introducing Xho I and EcoR I enzyme cutting sites at two ends of a nucleotide sequence, purifying, and connecting to a vector pET28a at 4 ℃ overnight under the action of T4 ligase, and naming the vector pET28 a-HCV; then the carrier is subjected to electric shock under the conditions of 7.5kV/cm of electric field intensity, 200 omega of resistance and 25 mu F of capacitance to be transformed into DH5 alpha competent cells; coating the DH5 alpha competent cells on an LB solid medium containing ampicillin, picking positive clones, inoculating the positive clones into an LB liquid medium, and culturing at 37 ℃ under shaking at 250rpm for overnight; the cells were collected by centrifugation at 3000rpm, and positive clones were identified by double digestion with Xho I and EcoR I using a plasmid extraction kit (purchased from Beijing complete gold Bio Inc.); the positive clone was sent to Shanghai bioengineering company for sequence identification, and the result showed that the sequence was correct.

The pET28a-HCV plasmid was digested with Xho I and EcoR I, purified and ligated overnight at 4℃under the action of T4 ligase to the vector prokaryotic expression vector pGEX 6P-1, which was transferred into E.coli BL 21. Inoculating the escherichia coli into an LB liquid culture medium, and performing shaking culture at 37 ℃ and 250rpm overnight to obtain seed liquid; inoculating 5% of seed solution into a 5L small-sized bioreactor, wherein the fermentation medium comprises 10g/L of tryptone, 5g/L of yeast extract, 2.34g/L of monopotassium phosphate, 3.58g/L of monopotassium phosphate, 10g/L of sodium chloride, 0.64g/L of magnesium sulfate heptahydrate, 0.56g/L of ferric chloride hexahydrate, 10g/L of glycerol and 5g/L of glucose (the balance is purified water); culturing at 37deg.C, introducing air at 0.8-1.5L/L.min, stirring at 350-450r/min until OD600nm reaches above 10, adding 0.2mM IPTG, inducing for 16 hr, and centrifuging to collect thallus.

Adding cell lysate (50 mmol/L sodium phosphate, 300mmol/L sodium chloride, 20mmol/L imidazole, pH 7.4) and 2mg/mL lysozyme, oscillating at 4deg.C for 30min, and performing ultrasonic treatment to fully break cells; then adding a cell lysate containing 6mol/L urea, carrying out ultrasonic dissolution of inclusion bodies in an ice bath, and carrying out oscillation action for 2 hours at 4 ℃; centrifugation was performed at 15000rpm for 30min at 4℃and the supernatant was collected. Protein was purified by Protein A method, the column was equilibrated with equilibration buffer (1 XPBS, 2mmol/L dithiothreitol, 0.1% sarcosyl, pH 7.4), and the Protein solution was eluted at a flow rate of 1mL/min to collect the target Protein. The renaturated sample was then collected by ultrafiltration with a 10kD retention, concentrated, sterilized by filtration through a 0.2 μm microporous membrane, and then subjected to SDS-PAGE electrophoresis (as shown in FIG. 2), and the gray value of the electrophoretic protein band was analyzed by Image J software to calculate the protein purity. Through detection, the purity of the protein reaches more than 97%, and the protein meets the requirement of subsequent experiments.

Example 2 screening and preparation of monoclonal antibodies targeting HCV

BALB/c female mice were selected 5, 6-8 weeks old. Female BALB/C mice were immunized with 50. Mu.g each, 3 times each at two week intervals, using the recombinant protein obtained in example 1. Serum from 20. Mu.L mouse tail venous blood was collected, serum titers of immunized mice were measured by indirect ELISA, and mice with higher relative titers were boosted by adjuvant-free intraperitoneal injection (50. Mu.g/mouse). After 1 week of the last immunization, when the number of B lymphoblastic cells was maximized, mouse spleen cells were taken for fusion.

Separating spleen cells into single cell suspension, mixing with mouse myeloma cells SP2/0 in logarithmic phase according to the ratio of 1:5, slowly dripping PEG, and performing cell fusion by using a cell fusion instrument; after fusion, cells were plated into 96-well cell culture plates, HAT screening reagent was added, and after 5 days of culture, a full change of fluid was performed. About 10 days of fusion, the 96-well culture supernatant is subjected to primary screening by adopting an indirect ELISA method, and ELISA positive hybridoma maternal clone cells are transferred into a 24-well cell culture plate for expansion culture.

BALB/C mice of 10-20 weeks of age were selected, and Freund's incomplete adjuvant (0.5 mL/mouse) was injected intraperitoneally in advance, 2X 10 mice each after 5-10 days ⁶ The individual cells can be collected after the abdominal cavity of the mouse is full of ascites, the ascites is purified by a Protein A method, and the antibody is quantitatively purified by SDS-PAGE detection (for specific methods, refer to section 1.2).

In this example, 10 monoclonal antibodies meeting the requirements were obtained by a total screening, and the affinity of the monoclonal antibodies to the target antigen was measured using a Fortebio biomacromolecule interactometer (available from eremoto corporation, usa) and the results are shown in table 1:

TABLE 1 affinity detection of individual antibodies to target antigens

Single domain antibodies	Affinity (nM)	Single domain antibodies	Affinity nM)
				01	6.33E-08	06	3.56E-09
02	7.45E-06	07	4.32E-06
				03	3.93E-07	08	5.46E-07
04	7.74E-08	09	1.62E-08
				05	7.36E-08	10	8.35E-09

In the invention, the 06 # antibody with higher affinity with the target antigen is selected for subsequent research so as to obtain the monoclonal antibody capable of efficiently recognizing HCV, and a corresponding detection reagent is prepared. Through sequence analysis, the heavy chain variable region of the No. 06 antibody comprises CDR-H1-3 with the amino acid sequence shown as SEQ ID No. 3-5; the light chain variable region comprises CDR-L1-3 with the amino acid sequence shown as SEQ ID No. 6-8. Further, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No. 9; the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID No. 10.

Example 3 preparation and testing of HCV detection kits

3.1 preparation of HCV detection kit

The hepatitis C virus detection kit is prepared by the embodiment and comprises the following components:

(1) Coated plate coated with hepatitis c virus antibody: 10mg of the monoclonal antibody provided in the present invention was coated in a 96-well plate;

(2) Chemiluminescent substrate: comprises a solution A and a solution B, wherein the solution A is 10mmol/L citric acid-sodium acetate buffer solution containing 0.6g/L carbamide peroxide; the substrate B solution is a 10mmol/L citric acid solution of 0.5g/L TMB (tetramethyl benzidine) and 0.6g/L EDTA-Na 2;

(3) Enzyme conjugate: the horseradish peroxidase is selected to mark the anti-human IgG monoclonal antibody;

(4) Positive control solution: is a diluted HCV antigen standard;

(5) Negative control solution: is a diluent without HCV antigen;

(6) Dilution liquid: 1.0mmol/L citric acid-sodium acetate buffer.

3.2 specificity experiments

In order to detect the detection specificity of the kit provided by the invention, ELISA is adopted to detect the kit in the embodiment, and the samples are blood samples respectively containing HAV (hepatitis A Virus), HBV (hepatitis B Virus), HCV (hepatitis C Virus), HIV (AIDS Virus) and Tubercle Bacillus (TB) infection, and the detection rate is calculated according to the positive result of detection, as shown in Table 2.

Table 2 test kit specificity test results

Virus species	Number of samples	Number of positives	Detection rate of
				HAV	82	3	3.66％
HBV	75	5	6.67％
				HCV	87	85	97.70％
HIV	67	1	1.49％
				TB	65	0	0.00％

The results show that the detection kit provided by the invention has high specificity for HCV and can effectively distinguish HCV from HAV, HBV, HIV, TB viruses.

3.3 sensitivity experiment

Serum was collected by centrifugation using clinical serum samples positive for HCV, diluted 10-fold, 100-fold, 1000-fold, 10000-fold, 100000-fold, and detected by ELISA using the kit, and the results are shown in table 3.

TABLE 3 sensitivity test results for detection kits

Sample of	1X 10 fold dilution	1×10 ² Dilution by multiple	1×10 ³ Dilution by multiple	1×10 ⁴ Dilution by multiple	1×10 ⁵ Dilution by multiple
						Detection result	Positive and negative	Positive and negative	Positive and negative	Negative of	Negative of

The experimental results show that the detection kit provided by the invention has higher sensitivity and can still effectively detect HCV positive blood samples when diluted by 1000 times.

3.4 stability test

The test kit was set at room temperature and tested for stability of positive or negative samples, and the results are shown in Table 4.

Table 4 test results of stability test kit

Room temperature standing time	For 1 week	For 2 weeks	4 weeks of	8 weeks of	For 12 weeks
						Positive sample	Positive and negative	Positive and negative	Positive and negative	Positive and negative	Positive and negative
Negative sample	Negative of	Negative of	Negative of	Negative of	Negative of

The results show that the kit provided by the invention has good detection stability, can still effectively detect the target sample after being placed at room temperature for 12 weeks, and can be stored for a longer time if being placed in an environment of 4 ℃.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A monoclonal antibody specifically recognizing HCV, wherein the heavy chain variable region of said antibody comprises CDR-H1-3 having the amino acid sequence shown in SEQ ID nos. 3-5; the light chain variable region of the antibody comprises CDR-L1-3 with an amino acid sequence shown as SEQ ID No. 6-8.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of said antibody is set forth in SEQ ID No. 9.

3. The monoclonal antibody of claim 1, wherein the amino acid sequence of the light chain variable region of said antibody is set forth in SEQ ID No. 10.

4. A kit for detecting hepatitis c virus comprising the monoclonal antibody of any one of claims 1-3.

5. Use of a monoclonal antibody according to any one of claims 1-3 for the preparation of a kit for detecting hepatitis c virus.

6. A preparation method of soluble HCV recombinant protein is characterized in that the amino acid sequence of the HCV recombinant protein is shown as SEQ ID NO.1, and comprises the following steps: constructing an expression vector carrying the HCV recombinant protein gene; introducing the expression vector into an E.coli expression system; culturing Escherichia coli by high-density fermentation; and (3) collecting escherichia coli thalli, and separating and purifying to obtain the soluble HCV recombinant protein.

7. The method of claim 6, wherein said HCV recombinant protein has a nucleotide sequence as set forth in SEQ ID No. 2.

8. The method of preparing a soluble HCV recombinant protein according to claim 7, further comprising: amplifying the recombinant protein gene by PCR, introducing Xho I and EcoR I enzyme cutting sites at two ends of a nucleotide sequence of the recombinant protein gene, connecting the recombinant protein gene to a pET28a vector after enzyme cutting, and electrically transforming the recombinant protein gene into DH5 alpha competent cells; screening positive clones, and performing enzyme digestion and sequencing to identify correct clones; connecting the recombinant protein gene to a prokaryotic expression vector pGEX 6P-1, introducing the prokaryotic expression vector into escherichia coli BL21, performing amplification culture, inoculating to a bioreactor, adding a fermentation medium, culturing at 37 ℃, and stirring at a speed of 350-450r/min, wherein the ventilation rate is 0.8-1.5L/L.min; adding IPTG to induce for 16 hours, and centrifugally collecting thalli; the thallus is crushed by ultrasonic, protein A is used for purifying Protein, and the soluble HCV recombinant Protein is obtained.

9. The method for producing soluble HCV recombinant protein according to claim 8, wherein said fermentation medium comprises 10g/L tryptone, 5g/L yeast extract, 2.34g/L potassium monohydrogen phosphate, 3.58g/L potassium dihydrogen phosphate, 10g/L sodium chloride, 0.64g/L magnesium sulfate heptahydrate, 0.56g/L ferric chloride hexahydrate, 10g/L glycerol, and 5g/L glucose.