CN116199774B - Monoclonal antibody for hepatitis B virus surface antigen mutant strain - Google Patents
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Abstract
The present application provides a monoclonal antibody directed against a hepatitis b virus surface antigen mutant. Screening and obtaining HBsAg antigen sequence of new mutant of hepatitis B virus from serum of hepatitis B patient, and introducing nucleotide sequence into colibacillus expression vector to prepare and obtain target antigen; immunizing an animal by utilizing the HBsAg antigen to obtain a corresponding monoclonal antibody, wherein the antibody can be combined with a target antigen with high specificity; by utilizing the monoclonal antibody, a related detection kit is prepared, and the kit has high sensitivity and specificity.
Description
Technical field:
the invention belongs to the field of disease diagnosis and detection, and particularly provides a monoclonal antibody for a hepatitis B virus surface antigen mutant strain.
The background technology is as follows:
hepatitis B Virus (HBV) infection is a global public health problem, and 15-25% of chronically infected persons develop severe disease and die prematurely due to cirrhosis, hepatocellular carcinoma or hepatitis. World health organization (The World Health Organization, WHO) estimated that of 2.4 million chronically HBV infected persons, about 780,000 deaths annually.
Currently, there is no specific antiviral therapeutic for patients with acute hepatitis b, about 95% of adults with normal infectious immunity will spontaneously recover, and the main goal of care is to keep comfortable, relieve symptoms and prevent the patient from infecting others. For chronic HBV infected persons, several therapeutic drugs and treatment regimens have now been provided, such as the U.S. food and drug administration has approved the treatment of chronic hepatitis b interferon- α (standard and pegylated) and oral antiviral drugs (entecavir, tenofovir, bipoxil fumarate, ilafenofovir and, less preferred, lamivudine, adefovir dipivoxil and tebifida). The current treatment of chronic hepatitis B can slow down or prevent the progress of liver cirrhosis, reduce the incidence of liver cancer, improve long-term survival rate and life quality, but can not cure. Thus, most patients who begin hepatitis B therapy must take medications for life, side effects of the therapy and the periodic monitoring required add to the difficulty and complexity of patient management. Thus, control of hepatitis b is largely dependent on early diagnosis and vaccination.
HBV is a DNA virus belonging to the family of hepadnaviridae, which was found by Blumerg doctor in 1965, and thus was found by Blumerg doctor to obtain the medical prize of Norbell in 1976. HBV virus, originally called Dane particle, is a 42 nm virus, HBV consisting of a nucleocapsid core surrounded by an outer lipoprotein shell (also called envelope). The virus contains 3 major structural antigens: surface antigens (Hepatitis B surface antigen, HBsAg), core antigens (Hepatitis B core antigen, HBcAg) and e antigens (Hepatitis B e antigen, HBeAg), wherein the HBsAg is expressed in large amounts and is present in the blood of infected individuals in the form of spherical and tubular particles (about 22 nm), making HBsAg an important target in the development of hepatitis b detection reagents and vaccines.
Because the clinical manifestation of hepatitis B cannot be clearly distinguished from other viral hepatitis, serological tests are required to be performed to clearly diagnose, and the tests use different (combined) serological markers to identify different stages of HBV infection, monoclonal antibodies are important means for detecting hepatitis B virus, and can specifically bind to target antigens to give accurate diagnosis results. However, with the use of hepatitis B vaccine and the natural evolution of virus, HBV mutants are endless, and the HBV content or infection in patients or blood products is difficult to effectively detect by using known monoclonal antibodies, which brings difficulty to clinical diagnosis and blood product management.
The invention separates and identifies new type hepatitis B virus mutant strain in the blood sample of hepatitis B patient, further obtains the HBsAg amino acid sequence, uses the HBsAg as target antigen, and screens and obtains monoclonal antibody, which can combine with target antigen with high specificity, has high sensitivity and specificity, and provides new solution for developing new type hepatitis B virus detection reagent.
Disclosure of Invention
In order to solve the technical problems, the invention provides a monoclonal antibody for resisting hepatitis B virus surface antigen, which comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1 with an amino acid sequence of SEQ ID NO. 1, LCDR2 with an amino acid sequence of SEQ ID NO. 2 and LCDR3 with an amino acid sequence of SEQ ID NO. 3; the heavy chain variable region comprises HCDR1 with the amino acid sequence of SEQ ID NO. 4, HCDR2 with the amino acid sequence of SEQ ID NO. 5, and HCDR3 with the amino acid sequence of SEQ ID NO. 6.
The monoclonal antibody provided by the invention can be combined with a target antigen with high specificity, and the antigen is from a novel hepatitis B virus mutant strain, so that the monoclonal antibody can effectively detect the hepatitis B virus mutant strain, and a solution is provided for researching and developing related detection reagents for detecting the qualification of blood products and diagnosing related virus infection conditions.
Further, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7.
Further, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8.
Provides a hepatitis B virus surface antigen, the amino acid sequence of which is shown as SEQ ID NO. 9.
The hepatitis B virus surface antigen is obtained by separating and identifying a hepatitis B patient blood sample from an inventor, has amino acid mutation at a corresponding site, and is difficult to effectively detect the mutant strain by adopting a known detection reagent, so that after the sequence analysis of HBsAg of the mutant strain, the HBsAg protein is prepared on a large scale by adopting an escherichia coli expression vector as a target antigen, and is used for screening novel monoclonal antibodies.
A kit for detecting hepatitis B virus is provided, which comprises the monoclonal antibody for resisting the hepatitis B virus surface antigen.
Further, the kit also comprises standard substance freeze-dried powder, sample diluent, a color reagent, a stop solution and a washing solution.
Further, the kit comprises a 96-well plate coated with the monoclonal antibody; horseradish peroxidase labeled anti-human IgG monoclonal antibody is used as an enzyme-labeled conjugate; the standard freeze-dried powder is hepatitis B virus surface antigen protein, and the amino acid sequence of the standard freeze-dried powder is shown as SEQ ID NO. 9; the sample diluent is PBS buffer solution with the concentration of 0.01mM and the pH value of 7.4; the solvent of the washing liquid is PBS buffer solution with 0.01mM of Tween-20 and pH value of 7.4 and the volume percentage concentration of Tween-20 is 0.2%; the stop solution is 2M sulfuric acid aqueous solution; the color reagent comprises horseradish peroxidase catalytic substrate A liquid and horseradish peroxidase catalytic substrate B liquid; the horseradish peroxidase catalytic substrate A solution is an H2O2 aqueous solution with the volume concentration of 3%; the horseradish peroxidase catalytic substrate B solution is a phosphate buffer solution containing 0.1mg/mL of 3,3', 5' -tetramethylbenzidine and having a concentration of 6.0 in 0.1mol/L, pH.
Provides a monoclonal antibody of the anti-hepatitis B virus surface antigen or application of the hepatitis B virus surface antigen in preparing a hepatitis B virus detection reagent.
Advantageous effects
The application provides a monoclonal antibody aiming at a hepatitis B virus surface antigen mutant strain and application thereof in preparing a hepatitis B detection reagent, and has the following advantages:
(1) Screening to obtain HBsAg antigen sequence of new mutant of hepatitis B virus, introducing nucleotide sequence into colibacillus expression vector, and preparing and obtaining target antigen;
(2) Immunizing an animal by utilizing the HBsAg antigen to obtain a corresponding monoclonal antibody, wherein the antibody can be combined with a target antigen with high specificity;
(3) By utilizing the monoclonal antibody, a related detection kit is prepared, and the kit has high sensitivity and specificity.
Drawings
Fig. 1: a HbsAg antigen nucleotide cleavage identification chart;
fig. 2: ROC curve;
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 identification of hepatitis B Virus mutant
Collecting 385 cases of hepatitis B patient blood samples, and centrifuging the patient blood 3000r for 10min to obtain serum; HBV DNA was extracted using the TIANamp Virus DNA/RNA Kit (available from TIANGEN company) and specific procedures were performed according to the Kit protocol.
The S fragment of the sample DNA is subjected to targeted amplification by nested PCR, and the amplification primer sequences are shown in Table 1.
TABLE 1 nested PCR primer sequences
The amplification reaction was performed using a nested PCR kit (purchased from TAKATA company), and the first round of PCR reaction system is shown in Table 2, and the reaction conditions are: 94 ℃ for 3min;94℃for 30s,58℃for 30s,72℃for 60s,30 cycles; and at 72℃for 5min.
TABLE 2 first round PCR reaction System
The second round PCR reaction system is shown in Table 3, and the reaction conditions are: 94 ℃ for 3min;94℃for 30s,58℃for 30s,72℃for 60s,30 cycles; and at 72℃for 5min.
TABLE 3 second round PCR reaction System
And (3) carrying out agarose gel electrophoresis on the amplified product to obtain a target band, and then sending the nested PCR product to Shanghai bioengineering company for gene sequencing. Different genotype reference sequences were downloaded from NCBI website and gene sequencing results were analyzed using website HIV sequence database (https:// www.hiv.lanl.gov/content/sequence/VESPA/VESPA. Html) sequence analysis tools and related bioinformatics software. A novel mutant of the HBsAg S region is identified, and the amino acid sequence of the mutant is shown as SEQ ID NO. 9.
Example 2HbsAg antigen preparation
The nucleotide sequence encoding the HBsAg antigen obtained in example 1 was synthesized, bamHI and XhoI restriction enzyme sites were introduced at both ends thereof, and after amplification and enrichment by PCR reaction, the amplified product was digested with the restriction enzymes BamHI and XhoI in a water bath at 37℃in a double digestion system: the target genes were 16. Mu.L, bamHI and XhoI were 1. Mu.L, and 10 Xbuffer was 2. Mu.L each. The ligation product was then purified using a nucleic acid purification kit (purchased from Shanghai Biotechnology Co., ltd.). The pET28a (+) plasmid was also double digested and purified in a similar manner using the restriction enzymes BamHI and XhoI. The double digested plasmid and gene were then ligated using T4 DNA Ligase (from Takara) in the following ligation system: 4 mu L of target gene, 4 mu L of plasmid, 1 mu L of T4 DNA Ligase and 1 mu L of 10 Xbuffer, and the ligation system was ligated at 4 ℃ overnight in a refrigerator to obtain plasmid vector pET28a (+) -HBsAg. The plasmid vector pET28a (+) -HBsAg after ligation was electrotransformed into BL21 (DE 3) competent cells, which were plated with ampicillin-containing LB agar medium, and cultured overnight in a constant temperature incubator at 37 ℃. Picking single colony, placing in LB liquid medium containing ampicillin, shake culturing at 37deg.C and 200rpm overnight, centrifuging at 3000rpm for 5min to collect thallus, extracting plasmid with plasmid extraction kit (purchased from Shanghai Biotechnology Co., ltd.), and enzyme cutting to identify and screen positive clone, and the result is shown in figure 1; positive clones were identified by gene sequencing and the nucleic acid sequence was correct and designated BL21 (DE 3) -HBsAg.
Activating BL21 (DE 3) -HBsAg strain, inoculating to LB culture medium containing antibiotics according to 5% inoculum size, shake culturing at 37deg.C and 200rpm for 4 hr, adding 0.5mM IPTG, reducing culture temperature to 25deg.C, and inducing culturing for 16 hr; centrifuging at 8000rpm for 5min, collecting bacterial mud, re-suspending bacterial mud with 1/10 volume of sterile PBS at 4deg.C, ultrasonic crushing, centrifuging at 10000rpm for 15min, filtering the supernatant with 0.45 μm filter membrane, separating with nickel ion affinity chromatographic column to obtain target protein antigen, and measuring to obtain protein with purity of above 95% which meets the requirement of subsequent experiment. The amino acid sequence of the obtained protein is shown as SEQ ID NO. 9 after sequencing.
EXAMPLE 3 screening and obtaining monoclonal antibodies
The purified HBsAg protein obtained in example 2 was used as an immunogen to prepare an immunogenic solution at a concentration of 50mg/mL, and 6-8 week old female Balb/C mice were subcutaneously multi-injected at a dose of 200. Mu.L/mouse, and three to four total immunizations were performed once a week. The serum antibody titer was determined by indirect ELISA method for tail vein blood collection of mice, and mice with high titer were selected for boosting by intraperitoneal injection of 200. Mu.L of HBsAg protein immunogen solution 3 days before cell fusion. The spleen cells of the immunized mice are collected aseptically, mixed with SP2/0 cells according to a certain proportion, and then subjected to cell fusion by PEG4000 according to a conventional method. Positive cell clones were screened using indirect ELISA by coating a 96-well plate with HBsAg protein, and 8 hybridoma cell lines stably secreting monoclonal antibodies were obtained, designated 1A2, 1A3, 2B5, 2C7, 3A2, 3F3, 5C5, 5D2, respectively.
Taking 6-8 week old healthy female Balb/c mice, and injecting sterilized liquid paraffin into the abdominal cavity of the female Balb/c mice with the age of 0.5 mL/mouse; after 7 days, each mouse was inoculated intraperitoneally with 1X 10 6 Each mL of the hybridoma cells was washed three times with serum-free medium before inoculation, and serum proteins were removed to avoid interference. After 7-10 days of injection, the abdomen of the mice is obviously swelled, and ascites is collected; after 3-5 days, the abdomen is inflated again and then collected continuously, and can be collected for 2-3 times generally. The ascites is collected in a centrifuge tube, centrifuged at 4000r/min for 0min, and the intermediate colorless transparent layer liquid is taken and the monoclonal antibody is purified by Protein A affinity chromatography.
The affinity of the antibody to the HBsAg protein is detected by using a protein interaction molecular dynamics detector ForteBio Octet Red e, HBsAg protein liquid with the concentration of 5 mug/mL is prepared, 200 mug of protein liquid is added into a biosensor, PBS is used as a control group, after reaction is carried out for 5min at 30 ℃, the binding condition of the antibody is detected, and the dissociation constant KD, the binding rate constant (Kon) and the dissociation rate constant (Koff) are calculated by software Octet Data Analysis Software.
The Tm values of the antibodies were measured using a thermal stability detector LightCycler480 II, the experimental parameters were: the excitation wavelength is 465nm, the emission wavelength is 580nm, and the temperature gradient is set to 25-95 ℃. The Acquisition Mode was set to Continuous, the rate of change was set to 0.01 ℃ per second, and the Acquisition (per ℃) was set to 50. The results are shown in table 4, and the 2B5 antibody having high affinity with the target antigen and good thermostability was selected for subsequent study and experiment. Through sequence identification and analysis, the antibody light chain variable region comprises LCDR1 with an amino acid sequence of SEQ ID NO. 1, LCDR2 with an amino acid sequence of SEQ ID NO. 2 and LCDR3 with an amino acid sequence of SEQ ID NO. 3; the heavy chain variable region comprises HCDR1 with the amino acid sequence of SEQ ID NO. 4, HCDR2 with the amino acid sequence of SEQ ID NO. 5 and HCDR3 with the amino acid sequence of SEQ ID NO. 6; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 7; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 8.
TABLE 4 antibody Performance detection
| Antibody numbering | Affinity KD value | Thermal stability Tm/. Degree.C |
| 1A2 | 6.22E-09 | 61.2 |
| 1A3 | 1.01E-10 | 63.2 |
| 2B5 | 2.23E-10 | 67.8 |
| 2C7 | 2.78E-09 | 57.3 |
| 3A2 | 4.88E-08 | 60.2 |
| 3F3 | 8.28E-09 | 69.1 |
| 5C5 | 4.31E-08 | 56.8 |
| 5D2 | 4.07E-08 | 64.5 |
Example 4 construction of detection kit and detection of hepatitis B Virus mutant Strain
4.1 construction of the detection kit
In order to effectively detect the mutant strain of the hepatitis B virus, the invention provides a detection kit, which comprises the monoclonal antibody against the surface antigen of the hepatitis B virus provided in the embodiment 3, and further comprises a standard lyophilized powder, a sample diluent, a color developing agent, a stop solution and a washing solution. The kit comprises a 96-well plate coated with monoclonal antibodies; horseradish peroxidase labeled anti-human IgG monoclonal antibody is used as an enzyme-labeled conjugate; the standard frozen powder is hepatitis B virus surface antigen protein, the preparation method is as shown in example 2, and the amino acid sequence of the standard frozen powder is shown in SEQ ID NO. 9; the sample diluent is PBS buffer solution with the concentration of 0.01mM and the pH value of 7.4; the solvent of the washing liquid is PBS buffer solution with 0.01mM of Tween-20 and pH value of 7.4 and the volume percentage concentration of Tween-20 is 0.2%; the stop solution is 2M sulfuric acid aqueous solution; the color reagent comprises horseradish peroxidase catalytic substrate A liquid and horseradish peroxidase catalytic substrate B liquid; the horseradish peroxidase catalytic substrate A solution is H with the volume concentration of 3% 2 O 2 An aqueous solution; the horseradish peroxidase catalytic substrate B solution is 0.1mol containing 0.1mg/mL of 3,3', 5' -tetramethyl benzidineL, pH is 6.0 phosphate buffer.
4.2 detection of hepatitis B Virus mutant
Collecting serum of 35 cases of hepatitis B patients, detecting 7 cases of hepatitis B virus mutant strains and 28 cases of known hepatitis B virus strains by nucleic acid, and detecting serum samples by ELISA method, wherein the method comprises the following specific steps: diluting a serum sample according to the ratio of 1:200, adding the serum diluent into a 96-well plate of the kit prepared in 4.1, and incubating overnight at 4 ℃; discarding the incubation liquid, adding the washing liquid, placing on a shaking table for washing for 10min at 100rpm, and washing for 3 times; after washing, adding the secondary antibody solution diluted according to the proportion of 1:200, and standing for 1h at room temperature; discarding the incubation liquid, adding the washing liquid, placing on a shaking table for washing for 10min at 100rpm, and washing for 3 times; after the washing is finished, the washing liquid is beaten and dried for 10min at room temperature; adding 100 mu L of color development liquid into each hole, and reacting for 15min at 37 ℃; the reaction was stopped by adding a stop solution, 50. Mu.L/well, and the OD was measured at 450 nm.
The experimental results were analyzed using GraphPad Prism 8 software, and ROC curves were drawn, as shown in fig. 2, with a specificity of 85.71%, a sensitivity of 64.57% and an AUC of 0.8112 for detecting the hepatitis b mutant virus strain, which can meet the detection requirements.
Claims (8)
1. A monoclonal antibody of anti-hepatitis B virus surface antigen comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises LCDR1 with an amino acid sequence of SEQ ID NO. 1, LCDR2 with an amino acid sequence of SEQ ID NO. 2 and LCDR3 with an amino acid sequence of SEQ ID NO. 3; the heavy chain variable region comprises HCDR1 with the amino acid sequence of SEQ ID NO. 4, HCDR2 with the amino acid sequence of SEQ ID NO. 5, and HCDR3 with the amino acid sequence of SEQ ID NO. 6.
2. The monoclonal antibody against hepatitis b virus surface antigen according to claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID No. 7.
3. The monoclonal antibody against hepatitis b virus surface antigen according to claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8.
4. A hepatitis B virus surface antigen has an amino acid sequence shown in SEQ ID No. 9.
5. A kit for detecting hepatitis b virus comprising the monoclonal antibody against hepatitis b virus surface antigen according to any one of claims 1 to 3.
6. The hepatitis b virus detection kit of claim 5, wherein: the kit also comprises standard substance freeze-dried powder, sample diluent, a color reagent, a stop solution and a washing solution.
7. The hepatitis B virus detection kit according to claim 6, wherein: the kit comprises a 96-well plate coated with the monoclonal antibody; horseradish peroxidase labeled anti-human IgG monoclonal antibody is used as an enzyme-labeled conjugate; the standard freeze-dried powder is hepatitis B virus surface antigen protein, and the amino acid sequence of the standard freeze-dried powder is shown as SEQ ID NO. 9; the sample diluent is PBS buffer solution with the concentration of 0.01mM and the pH value of 7.4; the solvent of the washing liquid is PBS buffer solution with 0.01mM of Tween-20 and pH value of 7.4 and the volume percentage concentration of Tween-20 is 0.2%; the stop solution is 2M sulfuric acid aqueous solution; the color reagent comprises horseradish peroxidase catalytic substrate A liquid and horseradish peroxidase catalytic substrate B liquid; the horseradish peroxidase catalytic substrate A solution is H with the volume concentration of 3% 2 O 2 An aqueous solution; the horseradish peroxidase catalytic substrate B solution is a phosphate buffer solution containing 0.1mg/mL of 3,3', 5' -tetramethylbenzidine and having a concentration of 6.0 in 0.1mol/L, pH.
8. Use of the monoclonal antibody against hepatitis b virus surface antigen according to any one of claims 1 to 3 or the hepatitis b virus surface antigen according to claim 4 for the preparation of a hepatitis b virus detection reagent.
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|---|---|---|---|---|
| KR20040058606A (en) * | 2002-12-27 | 2004-07-05 | 주식회사유한양행 | A humanized antibody against s-surface antigen of hepatitis b virus and a method for preparing thereof |
| CN1700010A (en) * | 2005-06-23 | 2005-11-23 | 北京热景生物技术有限公司 | Hepatitis B virus large protein pre-S1 antigen detection reagent kit |
| CN102492660B (en) * | 2011-11-28 | 2013-12-11 | 浙江大学 | Hepatitis B virus mutant, mutation amplification kit and application thereof |
| CN104662041A (en) * | 2012-09-27 | 2015-05-27 | 克鲁塞尔荷兰公司 | Human binding molecules capable of binding to and neutralizing hepatitis b viruses and uses thereof |
| WO2014193122A1 (en) * | 2013-05-31 | 2014-12-04 | (주)셀트리온 | Binding molecule able to neutralise hepatitis b virus |
| KR101839101B1 (en) * | 2014-11-28 | 2018-03-15 | (주)셀트리온 | Epitopes of Hepatitis B virus surface antigen and a binding molecule able to neutralize Hepatitis B virus specifically binding to epitopes thereof |
| CN107648602B (en) * | 2017-10-23 | 2020-09-01 | 苏州大学 | Bivalent hepatitis B vaccine and preparation method thereof |
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