CN111718397A - Screening and Recognition of CIM-ScFv Antibody Peptide Sequence Based on Murine Natural Single Chain Antibody Library and Its Application - Google Patents

Abstract

Screening and application of the polypeptide sequence of CIM-ScFv antibody based on the natural single-chain antibody library of murine origin and its application, the amino acid sequence of the heavy chain variable region of the polypeptide sequence is the sequence shown in SEQ ID NO. The amino acid sequence is shown in SEQ ID NO.2: QNGHSFPYT, the application of the polypeptide sequence in the identification of cimaterol antigen. The invention evaluates the quality of the obtained single-chain antibody, and the phage recovery rate has been increasing in the four rounds of screening, indicating that with the increase of the number of panning, the specific phage antibody is effectively enriched, and the cimaterol antigen Affinity identification is carried out, the affinity is good, and it can be used in the production of rapid detection reagents for cimaterol antigen, enriching the residual detection methods of cimaterol and other β2-type receptor agonists, in order to further prepare cimaterol antibodies and antibody evolution to lay a material foundation for the control and use of illegal additives such as cimaterol, so as to meet the public's requirements for residue-free animal meat products.

Description

Screening and Recognizing Peptide Sequences of CIM-ScFv Antibodies Based on Murine Natural Single Chain Antibody Library and its application

technical field

The invention belongs to the field of biotechnology, in particular to the screening and identification of a polypeptide sequence of a CIM-ScFv antibody based on a murine natural single-chain antibody library and its application.

Background technique

Cimaterol (CIM), which is often used by illegal breeding companies as a new "clenbuterol" instead of crotren. It has been demonstrated that CIM drug residues in animal edible tissues (such as lung, liver, kidney and muscle, etc.) pose potential health risks to consumers. The use of CIM as a growth promoter for livestock has been banned in many countries and regions, but misuse of CIM in order to increase economic profits is still common everywhere. In order to help solve food safety problems and improve the phenomenon that illegal additives are banned repeatedly in my country, and continue to improve the drug residue monitoring system, this study screened and identified the polypeptide sequences of CIM-ScFv antibodies based on the mouse-derived natural single-chain antibody library, aiming to enrich the The residual detection method of β2 receptor agonists such as cimaterol provides raw materials for the preparation of immunological detection reagents for cimaterol veterinary drug residues, and lays a scientific foundation for the development of recombinant antibodies to other small molecule haptens , to lay a material foundation for the further preparation of cimaterol antibodies and antibody evolution, and to provide a reference for the controlled use of cimaterol and other illegal additives to meet the public's requirements for residue-free animal meat products.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a polypeptide sequence that recognizes CIM-ScFv antibody based on the screening of mouse-derived natural single-chain antibody library and its application. The present invention firstly prepares a mouse-derived monoclonal antibody library, in order to screen out the anti-cimaterol recombinant antibody from it, Through the competitive inhibition ELISA standard curve results of soluble ScFv, it is shown that the phage antibody with the polypeptide sequence has a good degree of fit with the corresponding target antigen in the range of 3-243 ng/mL. , which can be used for quantitative and qualitative rapid detection of cimaterol drug residues.

To achieve the above object, the present invention adopts the following technical solutions:

Screening and recognizing the polypeptide sequence of CIM-ScFv antibody based on murine natural single-chain antibody library, it is characterized in that: the amino acid sequence of the heavy chain variable region of the polypeptide sequence is the sequence shown in SEQ ID NO.1: ARWTVITYAMDY, light chain The amino acid sequence of the variable region is shown in SEQ ID NO. 2: QNGHSFPYT.

Screening and identification of polypeptide sequences of CIM-ScFv antibodies based on murine natural single-chain antibody library in identification of cimaterol antigen.

The application of the polypeptide sequence recognizing CIM-ScFv antibody in the mouse-derived natural single-chain antibody library in the rapid detection of cimaterol antigen.

Use of screening and recognizing polypeptide sequences of CIM-ScFv antibodies based on a library of murine natural single-chain antibodies in the preparation of a kit for detecting cimaterol residues in a sample.

Further preferably, the sample includes, but is not limited to, animal tissue, urine and feed.

Further, the kits include but are not limited to ELISA kits.

Application of screening and recognizing polypeptide sequences of CIM-ScFv antibodies in immune-related diagnostic products based on murine natural single-chain antibody library.

More preferably, the immune-related diagnostic products include but are not limited to WB kits, ELISA kits, colloidal gold reagent strips and fluorescent diagnostic reagents

Compared with the prior art, the present invention has the following features and beneficial effects

The sequence shown in SEQ ID NO.1 provided by the present invention: ARWTVITYAMDY, and the sequence shown in SEQ ID NO.2: QNGHSFPYT, have good specificity, and can quickly synthesize cimaterol antibody according to the above sequence, and realize rapid artificial synthesis.

In the present invention, primers are first designed to amplify the variable region of mouse antibody gene, and the single-chain antibody is synthesized to construct recombinant phagemid pCANTAB-5E-ScFv, which is electrotransformed into host bacteria TG1, and rescued by helper phage M13K07 to obtain mouse-derived phage The single-chain antibody library, after identification, took the complete antigen of cimaterol as the target, and after four rounds of screening, the quality of the obtained single-chain antibody was evaluated. With the increase of the number of selections, the specific phage antibodies are effectively enriched, and the affinity identification with the cimaterol antigen is good. The residual detection method of β2 receptor agonists such as Luo, etc., lays a material foundation for the further preparation of cimaterol antibodies and antibody evolution, and provides a reference for the control and use of illegal additives such as cimaterol, so as to meet the public’s need for no residues. Requirements for animal meat products.

Description of drawings

Figure 1 shows the four efficient enrichment diagrams of specific phage antibodies;

Figure 2 is an illustration of the results of mouse RNA electrophoresis;

Fig. 3 is the electrophoresis result diagram of VH and VL;

Figure 4 SOE-PCR splicing ScFv antibody gene diagram;

Figure 5 shows the electrophoresis results of ScFv amplification;

Fig. 6 is a graphical representation of the test result of determining the antibody library titer;

Figure 7 shows the results of SDS-PAGE detection of soluble ScFv;

Figure 8. Graphical representation of the results of ic- ELISA detection of soluble ScFv.

Detailed ways

In order to make the technical means, innovative features, achieved goals and effects of the present invention easy to understand and understand, the present invention is further described below.

The embodiments described herein are specific embodiments of the present invention, are used to illustrate the concept of the present invention, are illustrative and exemplary, and should not be construed as limiting the embodiments of the present invention and the scope of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the contents disclosed in the claims and the description of the present application, and these technical solutions include any obvious technical solutions to the embodiments described herein. replacement and modification of

In the present invention, total RNA is first extracted from fresh spleen tissue of unimmunized healthy BABL/c mice, and then total antibody variable region cDNA is amplified by RT-PCR. Using a DNA linker encoding pentapeptide (Gly ₄ Ser) ₃ , the purified products of VH and VL genes were assembled into a complete mouse ScFv antibody gene. Finally, the recombinant ScFv fragment was cloned into the phage pCANTAB-5E vector, electroporated into E. coli TG1, and reinfected with the helper phage M13KO7 to obtain a murine recombinant phage with a functional single-chain fragment variable region (ScFv). The antibody library, after identification, took the complete antigen of cimaterol as the target, and after four rounds of screening, the quality of the obtained single-chain antibody was evaluated, and the phage recovery rate of each round was plotted on the ordinate, as shown in Figure 1, The phage recovery rate has been increasing, indicating that with the increase of the number of panning, the specific phage antibodies have been effectively enriched, and a single colony was picked for expansion and culture, and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. The sequencing primers are pCANTAB-5E universal primers S1 and S6. After removing the restriction enzyme site and the protection base, the MIGT database predicts the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2 of the present invention. The soluble expression and properties of the screened 13-CIM-ScFv positive phage clones were identified. It was identified that the soluble ScFv antibody produced by the hybridoma cell line had a titer of about 1:320 to CIM, which has a high antibody titer. , the antibody subtype is IgG1, the antibody has high affinity, and can be used for the detection of cimaterol drug residues. The soluble ScFv antibody reacts specifically with the antigen CIM, but does not react with BSA and OVA, indicating that phage 13 (13-CIM-ScFv) Antibodies have good specificity for CIM.

The experimental content involved in the present invention is described in detail below:

1. Obtain a murine recombinant phage antibody library of single-chain fragment variable region (ScFv);

1.1 Extraction and identification of mouse RNA

A 6-8 week old healthy unimmunized mouse was selected and fixed on a clean wax dish with the abdomen facing up. After sterilizing the abdomen with alcohol cotton balls, prepare two sets of sterilized scissors and forceps, one of which is used to cut the skin, and the other is used to cut the peritoneum and remove the spleen. After taking out the spleen, use scissors to remove the adhering tissue, and quickly transfer the intact spleen to a mortar (autoclaved) pre-cooled at -80 °C. During the grinding process, a small amount of liquid nitrogen can be appropriately added to make the grinding more sufficient. Pay attention to low temperature during this process to prevent RNA degradation and damage gene integrity. RNA was extracted from the fully lysed spleen tissue according to the operating steps of the kit.

The extracted RNA was diluted 10 times with TE buffer or RNAase-free Water, and the diluted solution was preliminarily identified with a micro-protein nucleic acid analyzer. After repeated zero correction with TE buffer or RNAase-free Water as a blank sample, the Dry the contacts and wait for the detection, record the ratio of A260/A280 and the nucleic acid concentration; finally, take 5 μL of RNA diluent and mix it with 1 μL of 6×Loading buffer and load the sample, the voltage is 150 v, and after 15 minutes, the gel imager is used for UV imaging. View Results. When the intact RNA is subjected to agarose electrophoresis, two bands of 28S and 18S rRNA will appear, and the former is about twice as bright as the latter. The specific results are shown in Figure 2. It can be seen that the extracted RNA has good integrity and can be used for subsequent PCR. Amplification reaction.

1.2 Synthesis of the first strand of mouse cDNA

The identified complete RNA was prepared on ice according to the instructions of the reverse transcription kit to prepare the RT reaction solution. After mixing the reaction solution, set up the PCR reaction in the PCR. The cDNA after the reaction was directly used for the amplification of the target fragment, or stored. Reserve at -20°C.

1.3 Primer design and synthesis

Use NCBI and IMGT databases to download mouse antibody genes, use DNAMAN software to compare and analyze the nucleic acid sequences of mouse antibody variable regions, and use Primer 5.0 software to design specific primers to amplify BABL/c mouse heavy chain variable region (VH) , the light chain variable region (VL) gene.

1.4 Synthesis of VH and VL

Using the first strand of cDNA as the template, PCR amplification was used to synthesize VH and VL of the mouse gene, respectively, and the PCR reaction system was prepared. The VH or VL primers used for variable region amplification were mixed primers, which were calculated according to the degeneracy of the primers. The relative proportions of each primer in a mix of primers such that equal amounts of each unique primer represented by a degenerate base are used. The reaction solution was vortexed and mixed, and after a brief centrifugation, the PCR amplification reaction was carried out, and the PCR amplification products were identified by electrophoresis. The results are shown in Figure 3. Afterwards, the PCR amplification products were recovered. The size of the fragment is 432 bp (Figure A in Figure 3), and the size of the VL fragment is 312 bp (Figure B in Figure 3), which are consistent with the expected results and can be used for the synthesis of ScFv.

It can be seen from Table 1 that there is a 30bp complementary overlapping region between the VH upstream primer and the VL upstream primer, so the Splicing overlap extension polymerase chain reaction (SOE-PCR) method can be used, as shown in Figure 4, the above VH The VL recovery product was linked without primers (shown in SOE-P1 in Figure 4) to assemble into ScFv gene products, and then primers were added to amplify the ScFv antibody gene fragment (shown in SOE-P2 in Figure 4). Therefore, after a large number of amplification of the above VH and VL, they were recovered and purified by a gel recovery kit, and the full-length ScFv gene was synthesized by the SOE-PCR method. The results are shown in Figure 5. The fragment size is about 780 bp. Increase and recover, and the recovered product is mouse single chain antibody gene. Take a sterilized PCR micro-reaction tube to prepare a PCR reaction solution, and store the PCR amplification products at -20 °C for identification and construction of recombinant vectors.

1.5 Construction of phage antibody library

1.5.1 Preparation of recombinant vector pCANTAB-5E-ScFv

The purified ScFv gene was ligated with the vector pCANTAB-5E to construct a recombinant phage vector. After configuring the connection system and mixing, connect it in a low temperature water bath at 16 °C for 10-12 h. The ligation product was added to the TG1 electrocompetent cells, and the mixture was immediately transferred to a clean, dry, pre-cooled electroporation cup for electrotransformation.

Immediately after the electric shock, 1 mL of SOC medium (preheated at 37 °C, with higher nutrient content than ordinary medium) was added to the click cup by pipetting gently, transferred to a new shaking tube, and incubated at 37 °C at 200 rpm for 1 h. Rejuvenate the strain; centrifuge at 5000 rpm for 5 min, retain about 200 μL of supernatant, discard the rest, resuspend the cells and spread them on 2×YT-A solid medium (containing 100 μg/mL ampicillin). ), and then placed upside down in a constant temperature incubator at 37 °C for overnight incubation.

The next day, a single colony with a clear edge was picked from the plate and cultured in 5 mL of 2×YT-A liquid medium at 37 °C and 180 rpm overnight. Take 1 mL of the bacterial solution after shaking culture, discard the supernatant after centrifugation, resuspend the bacterial cells in 20 μL of sterile water, and use it as a reaction template for PCR identification. The primers were ScFvFor SfiI and ScFvRev Not1 , and the reaction system and reaction procedure were the same as those of SOE-PCR.

1.6 Determination of antibody library titers

(1) Dilute the rescued phage antibody library with 2×YT medium (or PBS buffer) as follows: 99 µL of 2×YT medium is added to 1 µL of antibody library stock solution (1:100 dilution), mix well . Take 10 µL of the above mixture into 90 µL of 2×YT medium, which is the phage solution after 1000-fold dilution, which is vaguely many orders of magnitude. Proper and accurate dilution is the key to a good gradient phenomenon.

(2) Take 10 µL from the dilutions of the order of magnitude 10 ² , 10 ⁴ , 10 ⁶ , and 10 ⁸ , add 90 µL of TG1 medium (OD600=0.4), and co-culture at 37 °C for 30 min (same as M13K07 titer determination). ), and then mixed with 3 mL of top agar, poured into 2×YT-KA plate, and counted the number of plaques on the plate the next day. At the same time, a negative control was set up, and 100 µL of log-phase TG1 medium without adding the antibody library was cultured in the same way. The number of colonies on the plate decreases with the increase of the dilution factor of the phage library. If there is no single colony on the TG1 plate without adding phage, the result is credible. If not, the plaques need to be re-picked to repeat the construction and rescue of phage.

Figure 6 shows the growth of the antibody library dilution in this experiment on the plate. After the antibody library was diluted by 10 ⁸ , the number of effective single colonies grown on the plate was 156. According to the titer calculation formula, the results showed that the titer of the phage ScFv antibody library obtained in this study was 1.56×10 ¹⁰ pfu.

2 Soluble expression and identification of anti-cimaterol single-chain antibody in Escherichia coli

2.1 Soluble expression of 13-CIM-ScFv

Pick a single colony with good separation and clear edges, inoculate it in 5 mL of 2×YT-A medium, and shake it at 30 °C at 250 rpm overnight. Culture to log phase at 30 °C with shaking at 250 rpm. IPTG was added to the final concentration of 0.25, 0.1, and 0.5 mmol/L, and the culture condition was 30 °C. Different induction conditions and different induction times were used. Fully resuspended in TBS, placed at -20 °C for 20 min, placed at room temperature for 20 min, 10,000 rpm, 20 min, transferred the supernatant to a new tube for use, and resuspended the bacterial cells with 1 mL of hypertonic extract solution (pre-cooled on ice). Vortex at room temperature for 10 min, place at 4 °C for 10 min, centrifuge at 10,000 rpm for 20 min, discard the supernatant, add an equal volume of pre-cooled hypotonic buffer, stir gently at low temperature for 30 min, then centrifuge at 4 °C, 10,000 rpm , 20 min, and retain the supernatant, which contains the ScFv protein secreted into the periplasm.

2.2 SDS-PAGE detection of soluble ScFv

After the samples were processed, SDS-PAGE electrophoresis was performed on the culture supernatants of phage-positive ScFv proteins and control bacteria under different induction conditions. The blank control had no bands, and the supernatant of the uninduced bacterial liquid had more impurity bands. The induced bacterial liquid showed protein bands around 30KDa, which were in line with the expected size and the bands were relatively single, without too many impurities. It indicated that there was soluble expression of anti-cimaterol ScFv antibody in the bacterial periplasm of Escherichia coli HB2151. It can be seen from Figure 7 that the optimal induction conditions are 0.1 mmol/L IPTG, 30 °C, 16 h. This induction condition was selected as the induction condition for mass production of 13-CIM-ScFv. The results indicated that the protein had no frameshift during expression.

2.3 Preliminary establishment of the competitive inhibition ELISA assay method for soluble ScFv

2.3.1 Determination of working concentration of purified antibody

The titer of the ScFv protein secreted into the periplasm was determined using an indirect ELISA method. Note: Dilute the periplasmic cavity-extracted antibody with the primary antibody diluent starting from 1:5. The secondary antibody was Anti-E-tag (HRP) antibody at 1:4000 dilution, and the negative control was pCANTAB-5E-HB2151. The dilution factor corresponding to the absorbance value of about 1.0 and the P/N ratio greater than 2.1 is the optimal concentration of ScFv protein for competitive inhibition ELISA.

The results showed that all the antibodies could react with the coated antigen. It shows that the single chain antibody has been correctly folded and expressed. As shown in Table 1, the OD ₄₅₀ reading of the negative well is greater than 2.1 times as a positive well, and the maximum dilution ratio corresponding to the positive well is the titer of the antibody. From this, it can be judged that the titer of the antibody to CIM is 1:320 about.

Table 1 Determination of soluble ScFv titer results by indirect ELISA.

Serum dilution 1:5 1:10 1:20 1:40 1:80 1:160 1:320 1:640 Negative control OD₄₅₀ 1.173 1.013 0.883 0.662 0.490 0.370 0.298 0.203 0.133

2.3.2 Establishment of standard curve of competitive inhibition ELISA and determination of sensitivity and minimum detection limit

Eight standard concentrations were selected to establish a standard curve, and the 50% inhibitory concentration of the standard substance on the coated antigen was taken as the half inhibitory concentration (IC50). The lower the IC50 value, the lower the inhibitory effect and the higher the sensitivity. The determination method of the lowest detection line is the concentration of the corresponding small molecule inhibitor when the inhibition rate is 90%, and the concentration value corresponding to the OD value can be calculated to be 3.98 ng/mL by bringing it into the standard curve.

Specifically, the soluble antibody was selected at a dilution ratio of 1:10 as the primary antibody. Through the logarithm of 6 standard concentrations (1, 3, 9, 27, 81, 243ng/mL) and their corresponding absorbance values, the established standard curve is y = -0.2561x +1.0358, and the fit R ² = 0.9848, as shown in Figure 8. The half inhibitory concentration IC50=101 ng/mL and the lowest detection limit LOD=3.16 ng/mL of the competitive ELISA method established with this phage antibody had a good fitting degree in the range of 1-243 ng/mL.

The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

SEQUENCE LISTING

<110> Xinxiang College

<120> Screening and recognition of polypeptide sequences of CIM-ScFv antibodies based on murine natural single-chain antibody library and its application

<130> 20200330

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 12

<212> PRT

<213> Artificial sequences

<400> 1

Ala Arg Trp Thr Val Ile Thr Tyr Ala Met Asp Tyr

1 5 10

<210> 2

<211> 9

<212> PRT

<213> Artificial sequences

<400> 2

Gln Asn Gly His Ser Phe Pro Tyr Thr

1 5

Claims (8)

1. The polypeptide sequence for screening and identifying the CIM-ScFv antibody based on the murine natural single-chain antibody library is characterized in that: the amino acid sequence of the heavy chain variable region of the polypeptide sequence is shown as a sequence in SEQ ID NO. 1: ARWTVITYAMDY, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2: QNGHSFPYT are provided.

2. The use of the polypeptide sequence of the CIM-ScFv antibody of claim 1 in the identification of a cimaterol antigen.

3. The use of the polypeptide sequence of the CIM-ScFv antibody of claim 1 for the rapid detection of a cimaterol antigen.

4. Use of the polypeptide sequence of the CIM-ScFv antibody of claim 1 in the preparation of a kit for detecting cimaterol residues in a sample.

5. Use according to claim 4, characterized in that: such samples include, but are not limited to, animal tissue, urine, and feed.

6. Use according to claim 4, characterized in that: such kits include, but are not limited to, ELISA kits.

7. Use of the polypeptide sequence of the CIM-ScFv antibody of claim 1 in an immune-related diagnostic product.

8. The use of claim 7, wherein: the immune related diagnostic products include but are not limited to WB kit, ELISA kit, colloidal gold reagent strip and fluorescent diagnostic reagent.

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