A hepatitis B core antigen single-chain antibody phage library and its construction and application

Abstract

The present invention belongs to the field of biochemical technology, and specifically discloses a hepatitis B core antigen single-chain antibody phage library and its construction and application. The present invention successfully amplifies VH and VL fragments by designing HBcAg single-chain antibody heavy chain variable region primers (VH) and light chain variable region primers (VL), and uses the amplified VH and VL fragments and the designed HBcAg single-chain antibody full-length primers to amplify and splice to obtain scFv DNA fragments. On this basis, phage library technology is used to successfully construct a HBcAg single-chain antibody library, and HBcAg single-chain antibodies with high specificity and high sensitivity are screened therefrom, and an HBcAg precise quantitative kit can be further developed based on this. The technology provided by the present invention is of great significance to the clinical detection of HBcAg.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to a hepatitis B core antigen single-chain antibody phage library and its construction and application. Background Art

[0002] The "two-and-a-half" test for hepatitis B is currently the most commonly used serum marker for detecting hepatitis B virus (HBV) infection. The two-and-a-half test items include: hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), hepatitis B e antigen (HBeAg), hepatitis B e antibody (HBeAb), and hepatitis B core antibody (HBcAb). Since hepatitis B core antigen (HBcAg) is in a non-exposed state in the serum (composed of HBV core protein and the nucleocapsid is wrapped by the outer membrane protein), the detection method is relatively complicated, so this test is generally not performed clinically. However, with the in-depth study of the pathological molecular mechanism related to hepatitis B virus infection of hepatocytes, the importance of HBcAg has become increasingly prominent. At present, the consensus in the hepatitis B virus community is that the expression of HBcAg is positively correlated with the activity of HBV cccDNA in the nucleus of hepatocytes. The quantitative detection of HBcAg can indicate whether the cccDNA in chronic hepatitis B patients has been cleared or functionally inactivated. Therefore, the development of accurate quantitative reagents for HBcAg detection is of great clinical significance.

[0003] Currently, clinical monitoring of pathogens is all based on the principle of antigen-antibody reaction. Developing highly specific HBcAg antibodies is a key task. However, a large portion of the commercial antibodies currently used for HBcAg can bind to HBeAg, resulting in false positive detection reactions in clinical patient serum. Summary of the invention

[0004] In view of the shortcomings of the prior art mentioned above, the purpose of the present invention is to provide a hepatitis B core antigen single-chain antibody phage library and its construction and application, to prepare a single-chain antibody library of HBcAg using phage library technology, to screen highly specific and highly sensitive HBcAg single-chain antibodies therefrom, and to provide new technical means for the detection of HBcAg and the development of precise quantitative kits.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides, in a first aspect, a hepatitis B virus core antigen single-chain antibody (HBcAg-scFv), comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL and the heavy chain variable region VH both comprise a framework region and a complementarity determining region;

[0006] The framework region of the light chain variable region VL includes VL-FR1, VL-FR2 and VL-FR3, and the complementarity determining region of the light chain variable region VL includes VL-CDR1, VL-CDR2 and VL-CDR3,

[0007] The amino acid sequence of VL-FR1 is shown in SEQ ID NO.19 or SEQ ID NO.31,

[0008] The amino acid sequence of the VL-FR2 is shown in SEQ ID NO.21 or SEQ ID NO.33,

[0009] The amino acid sequence of the VL-FR3 is shown in SEQ ID NO.23 or SEQ ID NO.35,

[0010] The amino acid sequence of the VL-CDR1 is shown in SEQ ID NO.20 or SEQ ID NO.32,

[0011] The amino acid sequence of the VL-CDR2 is shown in SEQ ID NO.22 or SEQ ID NO.34,

[0012] The amino acid sequence of the VL-CDR3 is shown in SEQ ID NO.24 or SEQ ID NO.36;

[0013] The framework region of the heavy chain variable region VH includes VH-FR1, VH-FR2 and VH-FR3, and the complementarity determining region of the heavy chain variable region VH includes VH-CDR1, VH-CDR2 and VH-CDR3,

[0014] The amino acid sequence of VH-FR1 is shown in SEQ ID NO.25 or SEQ ID NO.37,

[0015] The amino acid sequence of VH-FR2 is shown in SEQ ID NO.27 or SEQ ID NO.39,

[0016] The amino acid sequence of the VH-FR3 is shown in SEQ ID NO.29 or SEQ ID NO.41,

[0017] The amino acid sequence of the VH-CDR1 is shown in SEQ ID NO.26 or SEQ ID NO.38,

[0018] The amino acid sequence of the VH-CDR2 is shown in SEQ ID NO.28 or SEQ ID NO.40,

[0019] The amino acid sequence of the VH-CDR3 is shown in SEQ ID NO.30 or SEQ ID NO.42.

[0020] Furthermore, the amino acid sequences of the VL-FR1, VL-FR2, and VL-FR3 are shown as SEQ ID NOs.19, 21, and 23, respectively, and the amino acid sequences of the VL-CDR1, VL-CDR2, and VL-CDR2 are shown as SEQ ID NOs.20, 22, and 24, respectively; the amino acid sequences of the VH-FR1, VH-FR2, and VH-FR3 are shown as SEQ ID NOs.25, 27, and 29, respectively, and the amino acid sequences of the VH-CDR1, VH-CDR2, and VH-CDR2 are shown as SEQ ID NOs.26, 28, and 30, respectively.

[0021] Furthermore, the amino acid sequences of the VL-FR1, VL-FR2, and VL-FR3 are shown in SEQ ID NOs.31, 33, and 35, respectively, and the amino acid sequences of the VL-CDR1, VL-CDR2, and VL-CDR2 are shown in SEQ ID NOs.32, 34, and 36, respectively; the amino acid sequences of the VH-FR1, VH-FR2, and VH-FR3 are shown in SEQ ID NOs.37, 39, and 41, respectively, and the amino acid sequences of the VH-CDR1, VH-CDR2, and VH-CDR2 are shown in SEQ ID NOs.38, 40, and 42, respectively.

[0022] The second aspect of the present invention provides a primer pair for amplifying the single-chain antibody gene sequence described in the first aspect, and the sequences thereof are shown in SEQ ID NOs. 17 and 18.

[0023] The third aspect of the present invention provides a primer set for constructing a hepatitis B virus core antigen single-chain antibody phage library, comprising a first primer pair for amplifying the single-chain antibody light chain variable region (VL) and a second primer pair for amplifying the single-chain antibody heavy chain variable region (VH);

[0024] The first primer pair includes upstream primers VL-F1, VL-F2, VL-F3, VL-F4 and VL-F5, and downstream primers VL-R1 and VL-R2, and the sequences are shown in SEQ ID NOs. 1 to 7 respectively;

[0025] The second primer pair includes upstream primers VH-F1, VH-F2, VH-F3, VH-F4 and VH-F5, and downstream primers VH-F6, VH-R1, VH-R2 and VH-R3, and the sequences are shown in SEQ ID NOs. 8 to 16, respectively.

[0026] The fourth aspect of the present invention provides a method for constructing a phage library of single-chain antibodies against hepatitis B virus core antigen, comprising: using phage library technology, using the primer pair described in the second aspect and the primer group described in the third aspect to construct a phage library of single-chain antibodies against hepatitis B virus core antigen.

[0027] Furthermore, the construction method comprises the following steps:

[0028] S1. Preparation of cDNA: cells of animals immunized with hepatitis B virus core antigen are taken to extract total RNA, the total RNA is separated and purified to obtain total mRNA, and the total mRNA is reverse transcribed into cDNA;

[0029] S2. Preparation of heavy chain variable region VH and light chain variable region VL fragments: Pair the upstream primers and downstream primers described in the third aspect in pairs, and amplify the VH and VL fragments separately by PCR;

[0030] S3. Preparation of single-chain antibody scFv DNA fragments by splicing: using VH and VL fragments to perform a first round of PCR reaction, and using the first round of PCR reaction products and the primer pair described in the second aspect to perform a second round of PCR reaction to obtain scFv DNA fragments;

[0031] S4. Construction of single-chain antibody library: After double restriction digestion of scFv DNA fragment, connect it to the phage backbone plasmid, and obtain primary scFv recombinant phage through phage display; use HBcAg recombinant protein to perform multiple rounds of panning on the primary scFv recombinant phage to obtain scFv phage that specifically binds to HBcAg, and obtain the hepatitis B virus core antigen single-chain antibody phage library.

[0032] Furthermore, in step S1, the cells are spleen cells of mice immunized with hepatitis B virus core antigen.

[0033] Furthermore, in step S2, the PCR reaction procedure is: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 15 seconds, annealing at 52°C for 15 seconds, extension at 72°C for 20 seconds, 30 cycles; extension at 72°C for 5 minutes; and insulation at 10°C.

[0034] Furthermore, in step S3, the VH fragments with positive amplification bands are purified and mixed at equimolar concentrations to obtain VH mixture, and the VL fragments with positive amplification bands are purified and mixed at equimolar concentrations to obtain VL mixture; then, VH mixture and VL mixture are mixed at equimolar concentrations to obtain VH / VL mixture, and then the first round of PCR reaction is performed.

[0035] Furthermore, in step S3, the first round of PCR reaction procedure is: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 20 seconds, 5 cycles; extension at 72°C for 5 minutes; and insulation at 4°C.

[0036] Furthermore, in step S3, the second round of PCR reaction procedure is: 95°C pre-denaturation for 2 minutes; 95°C denaturation for 15 seconds, 52°C annealing for 15 seconds, 72°C extension for 30 seconds, 30 cycles; 72°C extension for 5 minutes; 4°C insulation.

[0037] Furthermore, in step S4, the scFv DNA fragment is double-digested with EcoR I and Hind III restriction endonucleases.

[0038] Furthermore, in step S4, the phage backbone plasmid is a T7 phage backbone plasmid, and the phage is a T7 phage.

[0039] Furthermore, in step S4, after the scFv DNA fragment is double-digested with enzymes, the scFv DNA double-digested purified product is ligated with the phage backbone plasmid at a molar ratio of 3:1.

[0040] Furthermore, in step S4, the scFv DNA fragment is double-digested and then connected to the phage backbone plasmid to obtain the primary scFv recombinant phage through phage display, which includes:

[0041] After double restriction digestion, the scFv DNA fragment is connected to the phage backbone plasmid, and the connection product is mixed with the packaging extract for incubation; the packaging reaction mixture is added to the Escherichia coli host bacterial solution, cultured until flocs appear visible to the naked eye, and the culture is terminated when the bacterial solution becomes clear. After the bacterial solution is centrifuged, the supernatant is retained and added to the phage freezing solution and evenly mixed to obtain the primary scFv recombinant phage.

[0042] Furthermore, in step S4, the primary scFv recombinant phage is subjected to multiple rounds of panning using HBcAg recombinant protein to obtain scFv phage that specifically binds to HBcAg, and the hepatitis B virus core antigen single-chain antibody phage library is obtained, including:

[0043] HBcAg recombinant protein, HBeAg recombinant protein and BSA are taken to prepare HBcAg coated wells, HBeAg coated wells and BSA coated wells on the coated ELISA plate respectively; primary scFv recombinant phage is added to the HBcAg coated wells, incubated, washed, sodium dodecyl sulfate (SDS) is added, cooled, centrifuged, the supernatant is sucked out and added to the Escherichia coli host bacterial solution, cultured until flocs are visible to the naked eye, and the culture is terminated when the bacterial solution becomes clear; the phage lysate is centrifuged and the supernatant is taken out and added to the HBeAg coated wells and BSA coated wells in turn, standing to remove the phages non-specifically bound to HBeAg and BSA, collecting the supernatant, and completing the first round of panning; repeating the panning steps of phage supernatant and HBcAg binding for multiple rounds to obtain scFv phage that specifically binds to HBcAg, and obtaining a hepatitis B virus core antigen single-chain antibody phage library. The fifth aspect of the present invention provides a hepatitis B virus core protein single-chain antibody library constructed according to the method described in the fourth aspect.

[0044] The sixth aspect of the present invention provides the use of the single-chain antibody as described in the first aspect and / or the primer pair as described in the second aspect and / or the primer group as described in the third aspect and / or the library as described in the fourth aspect in the detection of hepatitis B virus core antigen, wherein the use is for non-disease diagnosis or treatment purposes.

[0045] Furthermore, the detection reagent is a kit for quantitatively detecting hepatitis B virus core antigen.

[0046] The seventh aspect of the present invention provides a hepatitis B virus core antigen detection reagent, comprising the single-chain antibody as described in the first aspect.

[0047] As described above, the hepatitis B core antigen single-chain antibody phage library of the present invention and its construction and application have the following beneficial effects:

[0048] The present invention successfully amplifies VH and VL fragments by designing HBcAg single-chain antibody heavy chain variable region (VH) primers and light chain variable region (VL) primers, and uses the amplified VH and VL fragments and the designed HBcAg single-chain antibody full-length primers to amplify and splice to obtain scFv DNA fragments, and on this basis, uses phage library technology to successfully construct a HBcAg single-chain antibody library, and screens out high-specificity and high-sensitivity HBcAg single-chain antibodies from them, and can further develop an HBcAg precise quantitative kit based on this. The technology provided by the present invention has great significance for the clinical detection of HBcAg. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1The SDS-PAGE electrophoresis diagrams in the preparation and purification of HBcAg and HBeAg recombinant proteins in the embodiments of the present invention are shown. Among them, M is a protein marker, lanes 1 and 2 are whole bacterial proteins expressed by HBcAg self-induction, and lane 3 is a whole bacterial protein control without induced expression; lane 4 is whole bacterial protein expressed by HBeAg self-induction, and lane 5 is a whole bacterial protein control without induced expression; lane 6 is the purified HBcAg recombinant protein, and lane 7 is the purified HBeAg recombinant protein.

[0050] Figure 2 Shown as Total DNA ( Figure 2 A), VL PCR / VH PCR products ( Figure 2 B) SCFV PCR product ( Figure 2 C) Agarose gel electrophoresis image.

[0051] Figure 3 The figure shows the identification result of the HBcAg-scFv phage library in the embodiment of the present invention. Figure 3 A is an electron microscope image of plaques obtained on a double-layer culture medium after gradient dilution of the phage library. Figure 3 B is an electron micrograph of 10 randomly selected plaques. Figure 3 C is the agarose gel electrophoresis detection result of PCR products.

[0052] Figure 4 The results of protein immunoelectrophoresis detection in the embodiment of the present invention are shown in FIG. Figure 4 A) and ECL luminescence imaging experimental results ( Figure 4 B) Among them, Figure 4 In A, M is a protein marker, lane 1 is the whole bacterial protein expressed by scFv No.07 induced by IPTG, and lane 2 is the whole bacterial protein control without induction; lane 3 is the whole bacterial protein expressed by scFv No.30 induced by IPTG, and lane 4 is the whole bacterial protein control without induction; lane 5 is the purified recombinant scFv No.07 protein; Figure 4 In B, lane 1 is HBeAg protein, lane 2 is antigen protein containing HBcAg protein ARD domain, and lane 3 is HBcAg protein. DETAILED DESCRIPTION

[0053] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0054] The specific implementation process of the present invention is as follows:

[0055] 1. Primer Design

[0056] In order to clone the PCR product into the phage vector, EcoR I and Hind III restriction endonuclease sites were introduced into the upstream and downstream primers, respectively. Design heavy chain variable region (VH) primers (VH-F1, VH-F2, VH-F3, VH-F4, VH-F5 and VH-F6, VH-R1, VH-R2, VH-R3) and light chain variable region (VL) primers (VL-F1, VL-F2, VL-F3, VL-F4, VL-F5 and VL-R1, VL-R2), amplify scFv full-length primers VL-SOE-VH-F and VL-SOE-VH-R. Primer sequences are shown in Table 1:

[0057] Table 1 Primer sequences

[0058]

[0059]

[0060] S=G / C, R=G / A, K=G / T, M=A / C, Y=C / T, W=A / T, ​​H=A / C / T, B=C / G / T, V=

[0061] A / C / G, D=A / G / T, and N=A / T / G / C.

[0062] 2. Preparation and purification of HBcAg and HBeAg recombinant proteins

[0063] The inoculation loop was used to streak the Rosetta E. coli (containing pET28a(+) / HBcAg plasmid) onto the main LB solid medium containing 50 μg / ml kanamycin, and then cultured overnight at 37°C for 12 hours. A single colony was picked from the plate and placed into 100 ml of autoinduction medium (12 g / L tryptone, 24 g / L yeast extract, 8 ml / L glycerol, 5 g / L lactose, 0.15 g / L glucose, 0.17 mol / L KH2PO4, 0.72 mol / L K2HPO4, 2 mmol / L MgSO4, 0.4% aspartic acid, 50 g / L kanamycin), and cultured at 37°C with shaking and rotation at 220 rpm for 24 hours. Collect the bacterial solution and centrifuge at 4000rpm for 20 minutes, discard the supernatant and retain the bacteria, and resuspend the bacteria with lysis buffer (5mmol / L imidazole, 2mol / L urea, 50mmol / L Tris-HCl (pH8.0), 300mmol / L NaCl, 0.5% Triton-X 100, 1mmol / L PMSF, 5mmol / Lβ-mercaptoethanol). Ultrasonic disruption of bacteria at 200W power, ice bath for 10 minutes. Centrifuge the bacterial disruption mixture at 4℃, 12000rpm for 20 minutes, separate the supernatant, place it in a clean high-pressure centrifuge tube, centrifuge it again at 4℃, 12000rpm for 20 minutes, collect the supernatant and place it on ice for later use. Wash the Ni-NTA affinity chromatography column once with 5 column volumes of distilled water, and then equilibrate the chromatography column with 2 column volumes of lysis buffer for 10 minutes. The supernatant was added to penetrate the column at a speed of 6 seconds / drop, and the penetrated liquid was recovered and stored at 4°C. The column was washed 5 times with 5 column volumes of washing buffer (20mmol / L imidazole, 2mol / L urea, 50mmol / L Tris-HCl, 300mM NaCl), and finally the bound protein on the column was eluted with 5 column volumes of elution buffer (250mmol / L imidazole, 2mol / L urea, 50mmol / L Tris-HCl, 300mmol / L NaCl). The eluate was ultrafiltered and concentrated, and then further purified by molecular sieve column to obtain high-purity HBcAg recombinant protein. The purified protein sample was taken, and the target protein band was identified by 15% SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining.

[0064] Purification of HBeAg recombinant protein: Collect the cells after culturing Rosetta Escherichia coli (containing pET28a(+) / HBeAg plasmid) and purify them using the same method as HBcAg.

[0065] 3. HBcAg immunization of BALB / c mice

[0066] For the first immunization, 60 μg of HBcAg recombinant protein was mixed with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple points into female BALB / c mice aged 6-8 weeks. For booster immunization every 14 days, 30 μg of HBcAg recombinant protein was mixed with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple points. After four booster immunizations, blood from the corners of the mice's eyes was collected to separate serum. After the serum titer was tested by ELISA, the selected mice were subjected to shock immunization (50 μg HBcAg intraperitoneal injection) for use 3 days later.

[0067] 4. Serum ELISA

[0068] HBcAg diluent (2μg / ml HBcAg, 0.1mol / L Na2CO3) and HBeAg diluent (2μg / ml HBeAg, 0.1mol / L Na2CO3) were coated on ELISA plates, incubated at 4°C overnight, and blocked with 5% bovine serum albumin (BSA) at 37°C for 2 hours. Mouse serum was diluted 2-fold starting from 200-fold, with blank control (blank) as PBS and negative control (negative) as 200-fold dilution of negative serum from unimmunized mice.

[0069] 5. Preparation of cDNA

[0070] The eyes of mice were removed and killed 3 days after shock immunization. The whole blood was collected and the serum was separated and stored for later use. The spleen was completely removed and cut into small pieces. It was placed on a 200-mesh stainless steel sieve and gently squeezed. It was then rinsed with sterile PBS solution. The rinse solution was centrifuged at 1000rpm at room temperature for 5 minutes to collect the spleen cell pellet. The spleen cells were washed once with sterile PBS solution and resuspended by centrifugation to a total volume of 3ml. 6ml TRNzol-A and 3ml chloroform were added and fully inverted for 5 minutes. Centrifuged at 4℃ and 12,000rpm for 10 minutes, 2.5ml of the supernatant was taken, 2.5ml of isopropanol and 250μL of sodium acetate were added, inverted and mixed, and then placed on ice for 1 hour. Centrifuged at 12,000rpm for 20 minutes, the precipitate was washed twice with 70% ethanol and dried at 37℃, 50μL TE was added to dissolve and transferred to a 1.5ml centrifuge tube. Total RNA was quantified and detected by MOPS formaldehyde denaturation 1% agarose gel electrophoresis. According to the instructions of Oligotex mRNA Mini Kits, total RNA was separated and purified to obtain mRNA (50 μL).

[0071] mRNA reverse transcription cDNA: add 1μL oligo dT (500μg / ml), 1μL dNTPMix (10mmol / L), 10μL mRNA in a 1.5ml centrifuge tube, react at 65℃ for 5 minutes, and quickly cool on ice; then continue to add 5×First-Strand Buffer 4μL, 0.1M DTT 2μL, RNaseOUT.Recombinant RibonucleaseInhibitor 1μL in the centrifuge tube, heat at 37℃ for 2 minutes, add 1μL M-MLV RT, incubate at 25℃ for 10 minutes, then incubate at 37℃ for 60 minutes, and finally incubate at 70℃ for 15 minutes. The total cDNA obtained was stored at -80℃ for future use.

[0072] 6. Preparation of VH and VL fragments

[0073] The upstream primers VH-F1, VH-F2, VH-F3, VH-F4, VH-F5, and VH-F6 were paired with the downstream primers VH-R1, VH-R2, and VH-R3 to amplify the VH region; the upstream primers VL-F1, VL-F2, VL-F3, VL-F4, and VL-F5 were paired with the downstream primers VL-R1 and VL-R2 to amplify the VL region.

[0074] The PCR reaction system included: 25 μL of 2× PrimeStar polymerase mixture, 1 μL of each 10 μmol upstream and downstream primers, 4 μL of cDNA, and deionized water to make up the total volume of 50 μL.

[0075] PCR reaction program: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 15 seconds, annealing at 52°C for 15 seconds, extension at 72°C for 20 seconds, 30 cycles; extension at 72°C for 5 minutes; insulation at 10°C.

[0076] The PCR amplification products were detected by 1.5% agarose gel electrophoresis.

[0077] 7. Preparation of splicing of scFv DNA fragments

[0078] The VH fragments with positive amplification bands were purified and mixed at equimolar concentrations to obtain VH mixture, and the VL fragments with positive amplification bands were purified and mixed at equimolar concentrations to obtain VL mixture; then, the VH mixture and the VL mixture were mixed at equimolar concentrations to obtain a VH / VL mixture and quantified.

[0079] First round reaction: 2× PrimeStar polymerase mixture 10 μL, VH / VL mixture 100 ng, add deionized water to a total volume of 20 μL. PCR amplification program: 95℃ pre-denaturation for 2 minutes; 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 20 seconds, 5 cycles; 72℃ extension for 5 minutes; 4℃ incubation.

[0080] Second round of reaction: Take 10 μL of the first round of reaction product, 20 μL of 2× PrimeStar polymerase mixture, 2 μL of 10 μmol / L upstream primer VL-SOE-VH-F and downstream primer VL-SOE-VH-R, and add deionized water to a total volume of 50 μL. PCR amplification program: 95℃ pre-denaturation for 2 minutes; 95℃ denaturation for 15 seconds, 52℃ annealing for 15 seconds, 72℃ extension for 30 seconds, 30 cycles; 72℃ extension for 5 minutes; 4℃ insulation.

[0081] The PCR product was identified by 1.2% agarose gel electrophoresis. The DNA fragment with a molecular weight of about 750 bp was the scFv spliced ​​DNA. The PCR product was purified and stored at -20°C.

[0082] 8. Preparation of HBcAg-scFv phage library

[0083] The scFv spliced ​​DNA was digested with EcoR I and Hind III restriction endonucleases at 37°C overnight and then purified by gel recovery. The scFv double digestion purification product was connected with the T7 phage backbone plasmid at a molar ratio of 3:1 at 16°C overnight. The ligation product was evenly mixed with T7 packaging extracts, incubated at 22°C for 2 hours, and 270 μL LB liquid culture medium was added to terminate the reaction. The packaging reaction mixture was added to 50 ml BLT5403 host bacterial solution (OD 600 =1.0), shake culture at 37°C for 2 hours, take out and observe every half hour, and stop culture when floccules are visible to naked eyes and the bacterial solution becomes clear. Centrifuge the bacterial solution at 8000rpm for 10 minutes, keep the supernatant and add phage freezing solution (final concentration 10% glycerol, final concentration 50mM NaCl, final concentration 10mM Tris-HCl) and mix evenly to obtain the primary scFv recombinant phage.

[0084] Dissolve the HBcAg recombinant protein in PBS to a final concentration of 50 μg / L. Coat each well of the ELISA plate with 150 μL, incubate at 4°C overnight, wash 3 times with PBST, add 150 μL of 5% BSA solution, incubate at room temperature for 4 hours, wash 3 times with PBST and dry, and prepare HBeAg-coated wells and BSA-coated wells under the same conditions for use. Add 150 μL of primary scFv recombinant phage to the HBcAg-coated wells, incubate at room temperature for 20 minutes, wash 5 times with PBST, add 150 μL of 1% SDS to each well, incubate at 37°C for 5 minutes, aspirate and place on ice for 10 minutes, centrifuge at 4°C, 10,000g for 5 minutes, aspirate the supernatant and add it to 50 ml of BLT5403 host bacterial solution (OD 600 =1.0), shake and culture at 37°C until visible floccules appear and the bacterial solution becomes clear. The phage lysate was centrifuged at 8,000g for 10 minutes, and the supernatant was added to the HBeAg-coated wells and BSA-coated wells in turn. The wells were allowed to stand at room temperature for 20 minutes to remove the phages that non-specifically bind to HBeAg and BSA, and the supernatant was collected to complete the first round of panning. The panning steps of phage supernatant binding to HBcAg were repeated for 3 rounds, and finally the scFv phage that specifically binds to HBcAg, i.e., HBcAg-scFv phage, was obtained.

[0085] 9. Identification of HBcAg-scFv phage library

[0086] 0.6% agarose-LB liquid medium was sterilized at high temperature and placed in a 50°C water bath for later use. BLT5403 fresh bacterial liquid (OD 600 =1.0) and place in an ice bath for later use. Place the LB solid culture dish in a 37°C incubator for preheating. Take 100 μL of HBcAg-scFv phage and add LB liquid culture medium at 10 -2 The concentration was diluted in a gradient to a minimum of 10 -14 Take 100μL of each gradient dilution into a clean 15ml test tube, add 250μL of bacterial solution and mix well, then immediately add 3ml of 50℃ 0.6% agarose-LB liquid medium, shake gently and mix well, then quickly pour into LB solid culture dish and spread it flat to form an upper gel. After the surface solidifies, turn it upside down and place it in a 37℃ incubator to culture until plaques appear. Calculate the number of plaques on the LB culture dish corresponding to the lowest dilution.

[0087] Pick a single plaque gel block and dissolve it in 50μL TE solution, heat it at 65℃ for 10 minutes, centrifuge it at 12,000g for 10 minutes, take 2μL supernatant as DNA template, identify primers provided by the kit, PCR program: first stage, 95℃ pre-denaturation for 2 minutes; second stage, 95℃ denaturation for 15 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 30 seconds, 30 cycles; third stage, 72℃ extension for 5 minutes, 4℃ insulation. PCR products were identified by 1.2% agarose gel electrophoresis and sent for sequencing.

[0088] 10. Expression of recombinant scFv in E. coli

[0089] Positive signal phages were amplified by PCR using sequencing primers. The PCR products were sequenced by Beijing Qingdao Biotechnology Co., Ltd. The nucleotide sequences of two scFv positive clones with different restriction structures were obtained. The framework region (FR) and complementary determining region (CDR) of each sequenced mouse VL and VH gene were analyzed. The positive PCR products were digested with restriction endonucleases EcoR I and Hind III and inserted into the pET28a vector. The recombinant plasmid was induced to express in Escherichia coli Rosetta at 37°C for 3h with 0.1mmol / L isopropylthiogalactoside (IPTG). The recombinant scFv protein was purified with Ni-NTA affinity beads at 4°C and labeled with horseradish peroxidase (HRP). The enzyme-labeled scFv protein was used as an antibody to detect HBc protein by protein immunoelectrophoresis. The HBc protein was separated on a 12% SDS-PAGE gel and then electrotransferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 60min and incubated with horseradish peroxidase-labeled scFv at room temperature for 60min. After washing, the bands on the membrane were developed and observed using ECL luminescent solution.

[0090] 11. Experimental results and analysis

[0091] 11.1. Prokaryotic Expression of HBcAg and HBeAg

[0092] Both HBcAg recombinant protein and HBeAg recombinant protein were expressed in vitro, and the results of self-induced expression of the recombinant strain and protein purification were detected by SDS-PAGE electrophoresis. Figure 1 As shown, the purity of HBcAg and HBeAg was above 90%.

[0093] 11.2. Preparation of scFv DNA

[0094] Total RNA was extracted from spleen cells of positive immunized mice and detected by MOPS formaldehyde denaturation 1% agarose gel electrophoresis ( Figure 2A) was followed by further purification using an mRNA purification kit to obtain total mRNA for the reverse transcription step, which is considered to be more likely to yield high-yield cDNA. In this experiment, about half of the primers amplified the target fragments VL and VH ( Figure 2 B), and then the VL DNA mixed fragment and the VH DNA mixed fragment were connected by overlapping PCR to obtain scFv DNA ( Figure 2 C).

[0095] 11.3.HBcAg-scFv recombinant phage library

[0096] According to the instructions of the T7 phage selection system kit, the phage library obtained after multiple rounds of screening was gradiently diluted and plaques were obtained on the double-layer culture medium. According to the dilution multiple and the number of plaques, the titer of the HBcAg phage library was calculated to be 8.5×10 13 Pfu / ml Figure 3 A). Randomly pick out 10 plaques for PCR identification, and the positive result ( Figure 3 B) The target fragment size is about 800-900 bp, among which No. 1 and No. 5 have the same molecular weight position, No. 2, No. 3 and No. 4 have the same molecular weight position, No. 6, No. 7 and No. 8 have the same molecular weight position, and No. 9 and No. 10 have no positive amplification. The identified PCR positive products were sent for sequencing, and the sequence comparison results proved that they were all positive recombinant scFv fragments, containing the complete VL-Linker-VH sequence.

[0097] 11.4. scFv Amino Acid Sequence Analysis

[0098] 100 phage monoclones were randomly selected for sequencing analysis of the VL and VH regions, and the sequencing results were analyzed by igblast software for the amino acid sequences of the framework region (FR) and the complementary determining region (CDR) (Table 2). The sequences of the VL and VH regions with the highest repetition rates are shown in Table 2: The repetition rates of the FR and CDR regions of the VL and VH regions represented by phage No. 07 and No. 30 reached 14% (14 of 100 strains were identical) and 8% (8 of 100 strains were identical), respectively, and the sequences of the representative strains are shown in Table 2.

[0099] Table 2. FR and CDR sequence analysis of scFv

[0100]

[0101]

[0102] 11.5. Recombinant Expression and Application of scFv

[0103] The two scFv DNAs were cloned into the prokaryotic expression vector pET28a(+) for recombinant expression. One of them was successfully expressed in E. coli Rosetta, while the other failed to express. After purification by Ni-NTA affinity beads ( Figure 4 A), the recombinant scFv protein combined with HRP can be used as an antibody to distinguish HBc protein from HBeAg in western blot. At the same time, the experimental research results also showed that the recombinant scFv peptide has a specific affinity for HBc ( Figure 4 B).

[0104] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A single-chain antibody against hepatitis B virus core antigen, characterized in that: comprising a light chain variable region VL and a heavy chain variable region VH, wherein the light chain variable region VL and the heavy chain variable region VH both comprise a framework region and a complementarity determining region; The framework region of the light chain variable region VL includes VL-FR1, VL-FR2 and VL-FR3, and the complementarity determining region of the light chain variable region VL includes VL-CDR1, VL-CDR2 and VL-CDR3; The framework region of the heavy chain variable region VH includes VH-FR1, VH-FR2 and VH-FR3, and the complementarity determining region of the heavy chain variable region VH includes VH-CDR1, VH-CDR2 and VH-CDR3; The amino acid sequences of the VL-FR1, VL-FR2, and VL-FR3 are shown in SEQ ID NOs. 19, 21, and 23, respectively, and the amino acid sequences of the VL-CDR1, VL-CDR2, and VL-CDR2 are shown in SEQ ID NOs. 20, 22, and 24, respectively; the amino acid sequences of the VH-FR1, VH-FR2, and VH-FR3 are shown in SEQ ID NOs. 25, 27, and 29, respectively, and the amino acid sequences of the VH-CDR1, VH-CDR2, and VH-CDR2 are shown in SEQ ID NOs. 26, 28, and 30, respectively.

2. Use of the single-chain antibody according to claim 1 in the detection of hepatitis B virus core antigen, wherein the use is for non-disease diagnosis or treatment purposes.

3. A hepatitis B virus core antigen detection reagent, characterized in that: Comprising the single-chain antibody as claimed in claim 1.

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