A single-chain antibody against hepatitis C core antigen and its preparation method and application

Abstract

The present invention relates to a hepatitis C core antigen single-chain antibody and a preparation method and application thereof. The hepatitis C core antigen single-chain antibody includes a single-chain antibody scFv-05 and a single-chain antibody scFv-12; the amino acid sequence of the single-chain antibody scFv-05 is shown in SEQ ID NO.1; the amino acid sequence of the single-chain antibody scFv-12 is shown in SEQ ID NO.5. The present invention first constructs a hepatitis C core antigen immunized mouse, and then obtains a hepatitis C core antigen single-chain antibody against hepatitis C virus with a new amino acid sequence through phage single-chain antibody display library technology and multiple biological selections. Compared with traditional antibodies, the single-chain antibody has a small molecular weight and can form a functional antibody without glycosylation modification. After obtaining the coding gene sequence of the single-chain antibody, it can be directly heterologously expressed by prokaryotic cells, realizing large-scale preparation, and the production cycle is short and the cost is low.

Description

Technical Field

[0001] The present invention relates to a hepatitis C core antigen single-chain antibody and a preparation method and application thereof, belonging to the field of biology and immunology technology. Background Art

[0002] Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus belonging to the genus Hepatitis C virus of the family Flaviviridae. It is identified as the main pathogenic virus of non-A, non-B hepatitis. The virus is mainly transmitted through blood and blood products. After HCV infects the human body, about 80% of infected people will develop chronic hepatitis, and 10-20% of infected people will develop cirrhosis and liver cancer. HCV infection is the fourth most common infectious disease after hepatitis B, tuberculosis, and dysentery. Therefore, HCV infection is still a public health problem that urgently needs to be controlled.

[0003] In recent years, hepatitis C core antigen (HCV-cAg) detection is considered to be one of the important methods for HCV detection. HCV-cAg is composed of 191 amino acid residues and is a relatively conservative structural region in the HCV genome. In addition to having the function of assembling viral particles as a nucleocapsid protein, it also plays an important role in viral proliferation and pathogenesis, and is an important marker of HCV infection. In addition, HCV-cAg appears relatively early after infection with hepatitis C, almost at the same time as HCV RNA. Therefore, HCV-cAg can be used as an early detection indicator for HCV infection, effectively shortening the detection window period of HCV, reducing the risk of HCV transmission, and facilitating early detection of infected persons. HCV-cAg detection is an immunological detection method, and antibodies are one of the important factors affecting the specificity and sensitivity of immunoassay methods.

[0004] Traditional antibodies include polyclonal antibodies (pAb) and monoclonal antibodies (mAb), but both have certain disadvantages. For example, there are differences between batches in the preparation of polyclonal antibodies; the preparation of monoclonal antibodies is time-consuming, costly, and complex. Through phage display technology, recombinant antibodies with high affinity can be selected from the antibody library, and the coding sequence of the antibody can be obtained by sequencing.

[0005] Single-chain antibody (scFv) is one of the most common recombinant antibodies. It is the smallest unit that can maintain effective binding with an antigen. It is a recombinant protein expressed by splicing the variable regions of light and heavy chains of antibodies through a gene encoding a connecting peptide. Single-chain antibodies have the advantages of being easy to express in prokaryotes and undergo genetic engineering. They are currently widely used in medical diagnosis, disease treatment, environmental monitoring, and food safety analysis. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides a single-chain antibody against hepatitis C core antigen and a preparation method and application thereof.

[0007] The technical solution of the present invention is as follows:

[0008] A single-chain antibody against hepatitis C core antigen, comprising single-chain antibody scFv-05 and single-chain antibody scFv-12;

[0009] The amino acid sequence of the single-chain antibody scFv-05 is shown in SEQ ID NO.1;

[0010] The amino acid sequence of the single-chain antibody scFv-12 is shown in SEQ ID NO.5.

[0011] Preferably according to the present invention, the single-chain antibody scFv-05 consists of a heavy chain variable region, a flexible peptide segment and a light chain variable region, and the connection order is heavy chain variable region-flexible peptide-light chain variable region.

[0012] Further preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.3, and the flexible peptide is a short peptide with an amino acid sequence of (Gly4Ser)3.

[0013] Preferably according to the present invention, the single-chain antibody scFv-012 consists of a heavy chain variable region, a flexible peptide segment and a light chain variable region, and the connection order is heavy chain variable region-flexible peptide-light chain variable region.

[0014] Further preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.6, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7, and the flexible peptide is a short peptide with an amino acid sequence of (Gly4Ser)3.

[0015] The method for preparing the above-mentioned hepatitis C core antigen single-chain antibody comprises the following steps:

[0016] (1) constructing mice immunized with hepatitis C core antigen, extracting spleen RNA from immunized mice, reverse-transcribing and synthesizing cDNA, and using the cDNA as a template to amplify and assemble single-chain antibody genes;

[0017] (2) cloning the single-chain antibody gene in step (1) into a phage vector, transforming competent cells, and obtaining a phage single-chain antibody library through helper phage infection;

[0018] (3) Using HCV core antigen as coating source, solid phase screening method was used to enrich anti-HCV core antigen phage single-chain antibody, and the HCV core antigen single-chain antibody was obtained through phage-ELISA screening.

[0019] A gene encoding a single-chain antibody against hepatitis C core antigen, the nucleotide sequence of the gene encoding the single-chain antibody scFv-05 is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the single-chain antibody scFv-12 is shown in SEQ ID NO.8.

[0020] An expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the coding gene of the above-mentioned hepatitis C core antigen single-chain antibody.

[0021] The use of the above-mentioned hepatitis C core antigen single-chain antibody or the coding gene of the hepatitis C core antigen single-chain antibody or the expression box, recombinant vector, recombinant bacteria or transgenic cell line containing the coding gene of the hepatitis C core antigen single-chain antibody in the preparation of diagnostic and / or preventive reagents, diagnostic and / or preventive kits, and the use includes diagnosing and / or preventing hepatitis C virus infection.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention first constructs mice immunized with hepatitis C core antigen, and then obtains a new amino acid sequence of hepatitis C core antigen single-chain antibody against hepatitis C virus through phage single-chain antibody display library technology and multiple bio-panning. Compared with traditional antibodies, the single-chain antibody has a small molecular weight and can form functional antibodies without glycosylation modification. After obtaining the coding gene sequence of the single-chain antibody, it can be directly heterologously expressed by prokaryotic cells, realizing large-scale preparation, and the production cycle is short and the cost is low.

[0024] 2. The hepatitis C core antigen single-chain antibodies scFv-12 and scFv-05 prepared by the present invention can specifically bind to the hepatitis C core antigen, have high sensitivity and specificity, and can detect hepatitis C virus with a sensitivity of up to 2.5 pg / mL, and the positive compliance rate for clinical HCV patients reaches 100%, and the negative compliance rate reaches 100%. Therefore, the hepatitis C core antigen single-chain antibody prepared by the present invention can be used to prepare reagents for diagnosing and / or preventing hepatitis C virus infection, and can be used for screening HCV infected persons in clinical practice, and has important significance and value for the clinical detection application of hepatitis C. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The electrophoresis results of different gene fragments in Example 1;

[0026] In the figure, A is the total RNA of mouse spleen tissue; B is the VH and VL gene fragments; C is the VH-Linker gene fragment (VH-L); D is the VL-Linker gene fragment (VL-L); and E is the single-chain antibody gene fragment (scFv).

[0027] Figure 2 This is the SDS-PAGE picture of the purified single-chain antibody in Example 3. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The examples described below are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0029] The amino acid sequence of the (Gly4Ser)3 short peptide is: GGGGSGGGGSGGGGS.

[0030] Hepatitis C core antigen (HCV-cAg) is available from Shandong Labo Biotechnology Co., Ltd. with a purity of >95%.

[0031] Example 1: Construction of a phage single-chain antibody library against hepatitis C core antigen

[0032] 1. Mouse immunization

[0033] 7-week-old female BALB / C mice were immunized with HCV core antigen as follows:

[0034] 100 μg of HCV core antigen was mixed with an equal volume of Freund's complete adjuvant, and an oil-in-water emulsion was prepared by grinding. The mixture was injected into mice at multiple points subcutaneously for the first immunization. The booster immunization was performed three times every two weeks (14 days) according to the same method. The dose of HCV core antigen was halved for each booster immunization, and the adjuvant was changed to Freund's incomplete adjuvant. One week after the fourth immunization, the tail vein blood of the mice was collected to detect the serum titer.

[0035] A non-immunized mouse was used as a negative control.

[0036] 2. Determination of serum titer in immunized mice

[0037] Dilute HCV core antigen to 1 μg / mL with carbonate buffer and coat on 96-well ELISA plate at 4°C overnight. Block with 5% skim milk powder and block at 37°C for 2h. Dilute serum with phosphate buffer at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6 400, 1:12800, 1:25600, 1:51200, 1:102400. Negative control is non-immunized mouse serum, and blank control is phosphate buffer. Add 100 μL of the test serum to each well, incubate at 37°C for 1h, and wash the plate with phosphate buffer (containing Tween-20). Add 100 μL of HRP-labeled goat anti-mouse monoclonal antibody (Sino Biological) diluted 1:5000 to each well, incubate at 37°C for 0.5h, and wash the plate with phosphate buffer (containing Tween-20). TMB colorimetric reagent was added for color development, and the reaction was terminated with stop solution after 10 min, and the reading was performed using an enzyme reader (490 nm). After ELISA detection, the data were shown in Table 1 below (average value), and the serum titer reached 1:51200, and the immune effect was acceptable, so the next step of the experiment could be carried out.

[0038] Table 1 Antibody titers in sera of immunized mice

[0039]

[0040] 3. Amplification of single-chain antibody (scFv) genes

[0041] (1) Take the spleen tissue of the immunized mouse with a titer that meets the experimental requirements, grind it into powder with liquid nitrogen, and use the Trizol method to extract RNA. First, take 5 μL of RNA for agarose gel electrophoresis. The results are as follows Figure 1 As shown in A. Take another 1 μL RNA and detect the concentration and purity of RNA using an ultra-micro spectrophotometer.

[0042] (2) The RNA with qualified concentration and purity was reverse transcribed into cDNA using FastKing cDNA First-Strand Synthesis Kit (Genomic Degraded; Tiangen Biochemical Technology (Beijing) Co., Ltd.). The specific experimental steps are shown in the instruction manual.

[0043] (3) The cDNA was diluted 10 times with ultrapure water and used as a template for amplification of the light chain variable region (VL) and the heavy chain variable region (VH). PCR amplification was performed using VH-F / VL-F and VH-R / VL-R as primers to obtain VH and VL gene fragments; wherein the primer sequences are shown in Table 2, the amplification system is shown in Table 3, and the amplification program is shown in Table 4.

[0044] Table 2. Primers used to amplify single-chain antibody genes

[0045]

[0046]

[0047] Note: R=A / G; Y=C / T; M=A / C; K=G / T; S=C / G; W=A / T; H=A / C / T; B=C / G / T; V=A / C / G; D=A / G / T.

[0048] Table 3. cDNA amplification system

[0049] Element Dosage (μL) cDNA 1.0 VH-F / VL-F (10 μM) 1.0 VH-R / VL-R (10 μM) 1.0 2×Taq PCR Mix 12.5 <![CDATA[ddH2O]]> 9.5

[0050] Table 4. cDNA amplification reaction procedure

[0051] program temperature time Pre-denaturation 95℃ 5min transsexual 95℃ 50S annealing 58℃(VL) / 54℃(VH) 1min(VL) / 40S(VH) extend 72℃ 50S Further extension 72℃ 7min

[0052] After the reaction is completed, 5 μL of the reaction solution is taken and verified by agarose gel electrophoresis and recovered by a gel recovery kit. The results are as follows Figure 1 As shown in B.

[0053] (4) Using the VH and VL genes recovered from the gel as templates, and using Linker-F / VH-F and VL-R / Linker-R as primers, PCR amplification was performed to obtain VH-Linker and VL-Linker gene fragments; wherein the primer sequences are shown in Table 2, and the reaction system is shown in Table 5.

[0054] Table 5. VH and VL amplification systems

[0055] Element Dosage (μL) VH / VL 1 Linker-F / VH-F 1 VL-R / Linker-R 1 2×Taq PCR Mix 12.5 <![CDATA[dd H2O]]> 9.5

[0056] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 1 min 30 s, annealing at 58°C for 1 min 30 s, extension at 72°C for 1 min, and extension at 72°C for another 7 min, for a total of 22 cycles.

[0057] After the reaction is completed, 5 μL of the reaction solution is taken and verified by agarose gel electrophoresis and recovered by a gel recovery kit. The results are as follows Figure 1 C and Figure 1 As shown in D.

[0058] (5) Overlap extension PCR amplification of single-chain antibody scFv gene

[0059] This step is divided into two-step PCR. First, the purified VH-Linker and VL-Linker are subjected to primer-free PCR at a molar mass of 1:1. The reaction system is shown in Table 6.

[0060] Table 6

[0061] Element Dosage (μL) VH-Linker Equal VL-Linker Equal 2×Taq PCR Mix 12.5 <![CDATA[ddH2O]]> Make up to 25

[0062] PCR reaction program: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 1 min, 72°C extension for 1 min, and 72°C extension for another 10 min, for a total of 10 cycles.

[0063] After the PCR was completed, primers Sfi IF, Not IR, and 2×Taq PCR Mix (primer sequences are shown in Table 2) were added to the above reaction solution, and the PCR reaction was continued. The PCR program was: 95°C pre-denaturation for 5 min, 95°C denaturation for 40 s, 58°C annealing for 40 s, 72°C extension for 50 s, and 72°C extension for another 10 min.

[0064] After the reaction, 5 μL of the reaction solution was taken and verified by agarose gel electrophoresis, and then recovered and purified by a gel recovery kit. The results are as follows: Figure 1 As shown in E.

[0065] 4. Construction of phage single-chain antibody library

[0066] (1) Construction of the recombinant vector pCANTAB-5E-scFv

[0067] The single-chain antibody gene fragment and vector pCANTAB-5E after gel recovery and purification were first digested by SfiⅠ (NEB), and the digested fragments were purified and then digested for the second time by NotⅠ (NEB). The SfiⅠ digestion system is shown in Table 7.

[0068] Table 7. SfiⅠ digestion system

[0069] Element Dosage S i 1μL scFv / pCANTAB-5E 1μg / 5μg CutSmart Buffer 5μL <![CDATA[ddH2O]]> Make up to 50 μL

[0070] At 37°C, the vector was digested for 1h 40min and the gene fragment was digested for 3h.

[0071] The digested fragments were purified by a purification kit ( PCR Purification Kit) was used for purification, and the NotⅠ restriction enzyme digestion system is shown in Table 8.

[0072] Table 8. NotⅠ digestion system

[0073] Element Dosage NotI 1μL scFv / pCANTAB-5E 1μg / 3μg CutSmart Buffer 5μL <![CDATA[ddH2O]]> Make up to 50 μL

[0074] At 37°C, the vector was digested for 100 min and the gene fragment was digested for 3 h. The product after double digestion was recovered by gel chromatography ( The purified vector and gene fragment were connected by T4 DNA ligase (NEB) at 16°C for 10-12h. The connection system is shown in Table 9.

[0075] Table 9. Connection system

[0076] Element Dosage (μL) pCANTAB-5E cloning vector 1 scFv gene 3 T4 DNA Ligase Buffer (10×) 2 T4 DNA Ligase 1 <![CDATA[ddH2O]]> 13

[0077] (2) Electrotransformation of ligation products

[0078] The overnight ligation product was purified by a purification kit ( PCR Purification Kit) for purification. Take out E.coil TG1 competent cells from -80℃ freezer and add 3μL ligation product to each tube. Mix gently and immediately transfer the mixture to a pre-cooled electroporation cup for electroporation. Set the electroporation conditions to: 1800V, 5ms. Immediately after electroporation, add 1mL of 37℃ pre-heated SOC medium and culture at 37℃, 150rpm for 1h. Spread the cultured bacterial liquid on SOC-G solid medium (100μg / mL ampicillin) and do 10 -1 -10 -7 The library capacity was estimated by plate counting after dilution gradient and incubated at 37°C for 12-16 h. The estimated library capacity was 7.3×10 8 The colonies on the plate were washed with 2×YT-G (100 μg / mL ampicillin) liquid medium, mixed and then added with an equal volume of 40% glycerol. The mixture was stored at -80°C to form the primary antibody library.

[0079] (3) Construction of phage single-chain antibody library

[0080] Take a primary antibody library from -80℃ and add it to 50mL of 2×YT-G liquid culture medium. Cultivate at 37℃ and 180rpm shaking until OD 600is 0.4. Add an appropriate amount of M13KO7 to the bacterial solution at a ratio of MOI = 1:20 (bacteria: helper phage), let it stand for infection for 30 minutes at 37°C, and shake it slowly at 150rpm for 30 minutes. Transfer the above bacterial solution to a 50mL sterile centrifuge tube, centrifuge it at 8000rpm for 15 minutes, discard the supernatant, resuspend the bacterial solution in 50mL 2YT-AK (70μg / mL kanamycin, 100μg / mL ampicillin) liquid culture medium, and culture it at 37°C, 150rpm shaking overnight. The next day, divide the culture solution into centrifuge tubes, centrifuge it at 4°C, 10000rpm for 20 minutes. Transfer the supernatant to another centrifuge tube, add 1 / 5 volume of pre-cooled PEG / NaCL, mix well and let it stand for 2h (ice bath). Centrifuge it at 4°C, 10000rpm for 30 minutes, discard the supernatant, and add 1mL ice-cold PBS to resuspend the precipitate. Centrifuge at 4°C, 12000 rpm for 10 min, transfer the supernatant to another sterile 1.5 mL EP tube, which is the phage antibody library. The titer of the antibody library was measured to be 1.5 × 10 11 pfu / mL and stored at 4°C until use.

[0081] Example 2: Screening of single-chain antibodies against hepatitis C core antigen

[0082] 1. Enrichment of HCV core antigen single-chain antibody

[0083] Dilute the HCV core antigen with coating solution to 80 μg / mL, and coat 100 μL of HCV core antigen per well on a 96-well plate at 4°C overnight. Prepare 3 mg / mL BSA and coat 100 μL per well on a 96-well plate at 4°C overnight. Discard the coating solution, wash the plate three times with PBST for 2 minutes each time, and pat dry. Block each well with 200 μL of 3% BSA at 37°C for 2 hours, wash the plate three times with PBST for 2 minutes each time, and pat dry. Take out the phage antibody library constructed in Example 1 (1.5×10 10 pfu), first incubate with 3% BSA at room temperature for 40 minutes, add 100μL per well to a 96-well plate, and incubate at 37°C for 1 hour. Wash the plate 10 times with PBS and BST for 2 minutes each time, and pat dry. Add 100μL HCl-glycine (PH 2.2) solution to each well to elute the bound phage, gently shake at 37°C for 10 minutes, immediately add 100μL Tris-HCl (PH7.4) solution to neutralize, repeatedly blow gently, and transfer the phage in the well to a 2mL EP tube. Take 10μL for phage titer determination, and the remaining phages are expanded for culture.

[0084] The above steps are the first round of screening process, and the screened phages are used for the next round of screening. A total of four rounds of screening were performed, and the HCV core antigen coating concentrations were reduced to 60μg / mL, 40μg / mL, and 20μg / mL in the second, third, and fourth rounds, respectively; the number of washes after the second round of phage incubation was increased to 15 times each in PBS and PBST, and the number of washes in the third and fourth rounds was increased to 20 times.

[0085] 2. Screening of specific positive monoclonal phage

[0086] 90 single clones were selected from the plate coated after the fourth round of screening and cultured overnight. 20 μL of the cultured bacterial solution was inoculated into 5 mL of 2×YT-G liquid medium and cultured at 37°C with shaking at 180 rpm until the OD 600 At about 0.4, add an appropriate amount of helper phage M13KO7, let stand at 37℃ for 30min, and shake gently on a shaker for 30min. Centrifuge at 4℃, 8000rpm for 10min, remove the supernatant, resuspend the precipitate in 5mL 2×YT-G liquid culture medium, and culture at 37℃, 150rpm overnight. Centrifuge the culture medium at 4℃, 8000rpm for 10min, and take 80μL of each single clone for Phage-ELISA.

[0087] Dilute HCV core antigen to 10 μg / mL with carbonate buffer, and coat on 96-well ELISA plate at 100 μL / well, and coat overnight at 4°C. The next day, discard the coating solution and wash the plate three times with PBST. Add 200 μL 3% BSA to each well and incubate at 37°C for 2 hours. Take 80 μL phage supernatant, add 20 μL 3% BSA to react for 30 minutes. Discard the blocking solution and wash the plate three times with PBST. Use the above phage liquid as the primary antibody, add it to the well, and incubate at 37°C for 2 hours. Discard the primary antibody and wash the plate 5 times with PBST. Add the secondary antibody HRP-anti-M13 at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate 5 times with PBST, add 100 μL / well TMB colorimetric solution, and incubate at 37°C in the dark for 10 minutes. After color development, add 50 μL 2% H2SO4 to terminate the reaction. Measure the OD of each well using a microplate reader 450 The results showed that 12 phage antibodies reacted with the antigen, and the values ​​are shown in Table 10. Two phage antibodies were strongly positive, named scFv-05 and scFv-12, respectively. The plasmids were extracted and sequenced.

[0088] The amino acid sequence of the hepatitis C core antigen single-chain antibody scFv-05 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.4;

[0089] The amino acid sequence of the hepatitis C core antigen single-chain antibody scFv-012 is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.8;

[0090] Table 10, Positive phage antibody OD 450 value

[0091]

[0092] Example 3: Heterologous expression of single-chain antibody against HCV core antigen

[0093] 1. Construction of expression vectors of single-chain antibodies scFv-05 and scFv-12 against HCV core antigen

[0094] Plasmids containing hepatitis C core antigen single-chain antibodies scFv-05 and scFv-12 were artificially synthesized, and BamH I and Nde I restriction sites were added at both ends of the sequence. The pET-28a (+) vector and the synthetic plasmids containing scFv-05 and scFv-12 were double-digested using restriction endonucleases BamH I and Nde I, respectively. The enzyme digestion system is shown in Table 11.

[0095] Table 11. Enzyme digestion system

[0096]

[0097]

[0098] The digestion temperature was 37°C and the digestion time was 1 h 40 min. The digested and purified pET-28a(+) was ligated with the scFv-05 and scFv-12 genes respectively by T4 DNA ligase (NEB) at 16°C for 10-12 h. The ligation system is shown in Table 12.

[0099] Table 12. Connection system

[0100] Element Dosage (μL) pET-28a(+) 1 AMO-scFv 3 T4 DNA Ligase Buffer (10×) 2 T4 DNA Ligase 1 <![CDATA[ddH2O]]> 13

[0101] Take a tube of Trans T1 competent cells, add 10 μL of ligation product, mix well, and let stand on ice for 30 minutes. Heat shock at 42℃ for 90 seconds, and immediately put it on ice for 2 minutes. Add 800 μL of LB liquid culture medium and shake at 37℃, 170rpm for 1 hour. Centrifuge at 12000rpm for 1 minute, and resuspend the remaining 300 μL of culture medium in the precipitate. Take 100 μL and spread it on LB solid (50 μg / mL kanamycin) plate, and invert and culture at 37℃ for 14 to 16 hours. Pick a single colony for culture, extract the plasmid, and perform PCR and sequencing identification. Take the plasmid extracted from the positive monoclonal colony to transform the Escherichia coli BL21 (DE3) competent cells, and the transformation steps are as above. Pick a monoclonal colony from the transformation plate cultured overnight for culture, extract the plasmid, perform PCR and sequencing identification, and obtain recombinant strains expressing scFv-05 and scFv-12 respectively.

[0102] 2. Induced expression and purification of HCV core antigen single-chain antibody scFv-05 and scFv-12

[0103] The positive recombinant strain was inoculated into LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm until OD 600 At about 0.5, add IPTG to a final concentration of 1mM, induce expression at 16℃ for 24h, collect the cells by centrifugation at 5000g and 4℃, and wash the cells twice with phosphate buffer. Resuspend the cells with binding buffer, add lysozyme to the bacterial solution, and freeze and thaw three times. Ultrasonicate the cells on ice, set the power to 200W, work for 10s, and rest for 10s. Centrifuge at 4℃, 8000rpm for 30min, collect the supernatant, which contains soluble single-chain antibody protein.

[0104] Purify using nickel affinity chromatography. Rinse the column with ultrapure water for about 3 column volumes, and wash the column with binding buffer (20mM Tris pH8.0) for about 2 column volumes. Filter the supernatant through a 0.45μm filter membrane, add it to the Ni column, incubate at low temperature for a certain period of time, and add binding buffer to rinse the column for about 2 column volumes. Add washing buffer (20mM Tris pH8.0, 50mM imidazole) to rinse the column for about 2 column volumes. Finally, add elution buffer (20mM Tris pH8.0, 250mM imidazole) to elute the target protein and collect the eluate.

[0105] Use ion exchange chromatography to purify the protein, rinse the column with ultrapure water for about 5 column volumes; rinse the column with balancing solution (20mM Tris pH8.0) for about 5 column volumes (flow rate is 1.5mL / min). Dilute the protein eluted from the Ni column by 2 times with the balancing solution and load it. Rinse the column with the balancing solution until the baseline is balanced for at least 10 column volumes. Use the washing buffer to elute the impurities (20mM Tris pH8.0, 100mM NaCl), and finally use the eluent (20mM Tris pH8.0, 400mM NaCl) to elute the target protein (flow rate is 1.5mL / min). When the UV value begins to rise, start collecting the eluent. When the UV value drops to close to 0.1, stop receiving the eluent. The collected eluent is a single-chain antibody solution. The supernatant was concentrated using an ultrafiltration concentrator, and the concentration of the single-chain antibody solution was measured using Nanodrop to obtain hepatitis C core antigen single-chain antibodies scFv-05 and scFv-12, respectively.

[0106] The purification of HCV core antigen single-chain antibody scFv-05 and scFv-12 was detected by SDS-PAGE. Figure 2 shown.

[0107] Depend on Figure 2 It can be seen that the present invention successfully heterologously expressed and purified scFv-05 and scFv-12.

[0108] Example 4: Indirect ELISA to detect the activity of HCV core antigen single-chain antibody scFv-05 and scFv-12

[0109] Use carbonate buffer (PH 9.6) to dilute the purified hepatitis C core antigen to 1 μg / mL, coat it in a 96-well ELISA plate at 100 μL / well, attach the sealing strip, and coat it overnight at 4°C. The next day, wash the plate three times with PBST, block it with 200 μL / well of blocking solution (3% BSA), and block it at 37°C for 2h. The purified hepatitis C core antigen single-chain antibodies scFv-05 and scFv-12 were diluted in phosphate buffer (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400), add 100 μL of antibody to each well, and incubate at 37°C for 1h. Wash the plate 5 times with PBST, add 100 μL of HRP-labeled anti-His tag antibody (1:5000 dilution) to each well, and incubate at 37°C for 0.5h. Wash the plate 5 times with PBST, add 100 μL TMB colorimetric solution to each well, incubate at 37°C in the dark for 10 min, and add 50 μL stop solution to each well. Read the OD value on the microplate reader. 450 The results are shown in Table 13.

[0110] Table 13. Indirect ELISA to detect single-chain antibody titer

[0111]

[0112] Example 5: Labeling and pairing verification of hepatitis C core antigen single-chain antibodies scFv-05 and scFv-12

[0113] 1. Labeling of HCV core antigen single-chain antibody scFv-05 and scFv-12

[0114] Weigh 5 mg HRP and dissolve it in 1 mL distilled water; add 0.5 mL 0.06 M NaIO4 solution to the HRP solution and let it stand at 4 °C for 30 min; continue to add 0.5 mL 0.16 M polyethylene glycol and let it stand at room temperature for 30 min; add 5 mg purified hepatitis C core antigen single-chain antibody scFv-05 and scFv-12 respectively, mix well and transfer to dialysis bag, dialyze against 0.05 M pH = 9.5 carbonate buffer overnight; on the second day, add 0.2 ml 5 mg / mL NaBH4 solution, mix well and let it stand at 4 °C for 2 hours; slowly add an equal volume of saturated (NH4)2SO4 solution to the resulting solution, let it stand at 4 °C for 30 min, then centrifuge to remove the supernatant, and use 0.02 M Dissolve the precipitate with PBS buffer (pH=7.4) and transfer it into a dialysis bag. Desalt it with the same liquid at 4°C overnight. Centrifuge the next day and take out the supernatant to obtain HRP-scFv-05 and HRP-scFv-12. Add 0.02M pH7.4 PBS solution to 5mL. Add an equal amount of glycerol, divide the mixture into portions and store at low temperature for later use.

[0115] 2. Pairing verification of HCV core antigen single-chain antibody scFv-05 and scFv-12

[0116] Use carbonate buffer (PH 9.6) to dilute the purified hepatitis C core antigen single-chain antibody scFv-05 and scFv-12 to 2.5μg / mL, and coat them in a 96-well ELISA plate at 100μL / well. A sealing strip is attached and coated overnight at 4℃. The next day, wash the plate three times with PBST, block with 200μL / well blocking solution (3% BSA), and block for 2h at 37℃. Add different concentrations of hepatitis C core antigen (0pg / mL, 50pg / mL, 100pg / mL, 200pg / mL, 400pg / mL, 800pg / mL, 1600pg / mL), add 100μL of antigen to each well, and incubate at 37℃ for 1h. Wash the plate 5 times with PBST, add 100 μL of HRP-scFv-12 / HRP-scFv-05 (1:4000) obtained in the previous step to each well, and incubate at 37°C for 0.5 h. Wash the plate 5 times with PBST, add 100 μL of TMB colorimetric solution to each well, incubate at 37°C in the dark for 10 min, and add 50 μL of stop solution to each well. Read the OD value on the microplate reader 450 The results are shown in Table 14.

[0117] Table 14. Verification of the pairing of single-chain antibody scFv-05 and scFv-12

[0118]

[0119] As shown in Table 14, the hepatitis C core antigen single-chain antibody scFv-05 and scFv-12 can be paired with each other to detect the HCV-cAg concentration, but the detection effect of scFv-12 as a capture antibody and scFv-05 as a detection antibody is better than that of scFv-05 as a capture antibody and scFv-12 as a detection antibody.

[0120] Case study 6: Comparison of sensitivity of HCV core antigen single-chain antibody scFv-05 and scFv-12 with traditional monoclonal antibodies

[0121] 1. Sensitivity of HCV core antigen single-chain antibody scFv-05 and scFv-12

[0122] Use carbonate buffer (PH 9.6) to dilute the purified hepatitis C core antigen single-chain antibody scFv-12 to 2.5μg / mL, and coat it in a 96-well ELISA plate at 100μL / well, affix the sealing strip, and coat it overnight at 4℃. The next day, wash the plate three times with PBST, block it with 200μL / well of blocking solution (3% BSA), and block it at 37℃ for 2h. Add different concentrations of hepatitis C core antigen (0pg / mL, 1.25pg / mL, 2.5pg / mL, 5pg / mL, 10pg / mL, 20pg / mL, 40pg / mL, 80pg / mL, 160pg / mL, 320pg / mL, 640pg / mL), add 100μL of antigen to each well, and incubate at 37℃ for 1h. Wash the plate 5 times with PBST, add 100 μL HRP-labeled hepatitis C core antigen single-chain antibody scFv-05 (1:4000) to each well, and incubate at 37°C for 0.5 h. Wash the plate 5 times with PBST, add 100 μL TMB colorimetric solution to each well, incubate at 37°C in the dark for 10 min, add 50 μL stop solution to each well, and read the OD value on the microplate reader. 450 The numerical value.

[0123] 2. Sensitivity of traditional monoclonal antibodies

[0124] The hepatitis C virus core antigen detection kit (enzyme-linked immunosorbent assay) produced by Shandong Lab Biotechnology Co., Ltd. was used to detect different concentrations of hepatitis C core antigen (0pg / mL, 1.25pg / mL, 2.5pg / mL, 5pg / mL, 10pg / mL, 20pg / mL, 40pg / mL, 80pg / mL, 160pg / mL, 320pg / mL, 640pg / mL). 100μL of antigen was added to each well and the OD value of the microplate reader was read. 450 The test results are shown in Table 15.

[0125] Table 15. Sensitivity of single-chain antibody scFv and traditional monoclonal antibodies

[0126]

[0127] As shown in Table 15, the hepatitis C core antigen single-chain antibodies scFv-05 and scFv-12 prepared by the present invention have higher sensitivity than traditional monoclonal antibodies.

[0128] Implementation Case 7: Testing of clinical samples from patients infected with hepatitis C virus

[0129] Use carbonate buffer (PH 9.6) to dilute the hepatitis C core antigen single-chain antibody scFv-12 to 1μg / mL, and coat it in a 96-well ELISA plate at a volume of 100μL / well. Attach a sealing strip and coat it overnight at 4℃. The next day, wash the plate three times with PBST and block it with 200μL / well of blocking solution (3% BSA) at 37℃ for 2h. Add 100μL of serum sample to each well and incubate at 37℃ for 1h. Wash the plate 5 times with PBST, add 100μL of HRP-labeled hepatitis C core antigen single-chain antibody scFv-05 (1:4000) to each well, and incubate at 37℃ for 0.5h. Wash the plate 5 times with PBST, add 100μLTMB colorimetric solution to each well, incubate at 37℃ in the dark for 10min, add 50μL of stop solution to each well, and read the OD value of the ELISA reader. 450 The test results are shown in Table 16 below.

[0130] Table 16. Clinical sample testing

[0131]

[0132] The results showed that the detection antibodies prepared by the present invention were effective for:

[0133] Specificity: 200 / 200×100%=100%;

[0134] Sensitivity: 72 / 72×100%=100%;

[0135] Consistency: (72+200) / (72+200)×100%=100%.

[0136] It can be seen from Table 16 that, after verification by testing clinical samples, the hepatitis C core antigen single-chain antibodies scFv-12 and scFv-05 provided by the present invention have high sensitivity and specificity for the detection of hepatitis C core antigen, can be used to prepare reagents for diagnosing and / or preventing hepatitis C virus infection, and can be used for the screening of HCV-infected patients in clinical practice, which is of great significance and value for the clinical detection application of hepatitis C.

Claims

1. A single-chain antibody against hepatitis C core antigen, characterized in that: The hepatitis C core antigen single-chain antibody includes single-chain antibody scFv-05 and single-chain antibody scFv-12; The amino acid sequence of the single-chain antibody scFv-05 is shown in SEQ ID NO.1; The amino acid sequence of the single-chain antibody scFv-12 is shown in SEQ ID NO.

5.

2. The hepatitis C core antigen single-chain antibody according to claim 1, characterized in that The single-chain antibody scFv-05 is composed of a heavy chain variable region, a flexible peptide segment and a light chain variable region, and the connection order is heavy chain variable region-flexible peptide-light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.3, and the flexible peptide is a short peptide with an amino acid sequence of (Gly4Ser)3.

3. The single-chain antibody against hepatitis C core antigen according to claim 1, characterized in that The single-chain antibody scFv-012 is composed of a heavy chain variable region, a flexible peptide segment and a light chain variable region, and the connection order is heavy chain variable region-flexible peptide-light chain variable region; The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.6, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.7, and the flexible peptide is a short peptide with an amino acid sequence of (Gly4Ser)3.

4. A gene encoding a single-chain antibody against hepatitis C core antigen, characterized in that: The nucleotide sequence of the gene encoding the single-chain antibody scFv-05 is shown in SEQ ID NO.4; the nucleotide sequence of the gene encoding the single-chain antibody scFv-12 is shown in SEQ ID NO.

8.

5. An expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the gene encoding the hepatitis C core antigen single-chain antibody according to claim 4.

6. Use of the hepatitis C core antigen single-chain antibody according to claim 1 or the coding gene of the hepatitis C core antigen single-chain antibody according to claim 4 or the expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the coding gene of the hepatitis C core antigen single-chain antibody according to claim 5 in the preparation of a kit for diagnosing hepatitis C virus infection.

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