Polypeptide sequence for screening and identifying CIM-ScFv antibody based on murine natural single-chain antibody library and application thereof
阅读说明：本技术 基于鼠源天然单链抗体库筛选识别CIM-ScFv抗体的多肽序列及其应用 (Polypeptide sequence for screening and identifying CIM-ScFv antibody based on murine natural single-chain antibody library and application thereof ) 是由 王选年 岳锋 司艳芳 郭东光 齐永华 潘鹏涛 李鹏 孙国鹏 朱艳平 于 2020-05-19 设计创作，主要内容包括：基于鼠源天然单链抗体库筛选识别CIM-ScFv抗体的多肽序列及其应用,多肽序列的重链可变区的氨基酸序列为如SEQ ID NO.1所示序列：ARWTVITYAMDY,轻链可变区的氨基酸序列如SEQ ID NO.2所示序列：QNGHSFPYT,多肽序列在西马特罗抗原鉴定中的应用。本发明对获得的单链抗体进行质量评价,四轮筛选中噬菌体回收率一直呈增加的趋势,说明随着淘选次数的增加,特异性的噬菌体抗体得到了有效富,与西马特罗抗原进行亲和力鉴定,亲和力较好,可应用于西马特罗抗原的快速检测试剂的生产中,丰富西马特罗等β2型受体激动剂的残留检测方法,为进一步制备西马特罗的抗体和抗体进化奠定物质基础,为西马特罗等非法添加物的控制使用提供参考,以满足公众对无残留动物肉制品的要求。(The polypeptide sequence for screening and identifying the CIM-ScFv antibody based on the murine natural single chain antibody library and the application thereof, wherein the amino acid sequence of the heavy chain variable region of the polypeptide sequence is shown as the 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, application of the polypeptide sequence in cimaterol antigen identification. The invention carries out quality evaluation on the obtained single-chain antibody, the phage recovery rate in four-round screening is always in an increasing trend, which shows that the specific phage antibody is effectively enriched along with the increase of the elutriation times, the affinity identification is carried out on the phage antibody and the cimaterol antigen, the affinity is better, the invention can be applied to the production of a rapid detection reagent of the cimaterol antigen, the residue detection method of the cimaterol and other beta 2-type receptor agonists is enriched, a material basis is laid for further preparation of the cimaterol antibody and antibody evolution, a reference is provided for the control and use of cimaterol and other illegal additives, and the requirement of the public on residue-free animal meat products is met.)
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.
Technical Field
The invention belongs to the technical field of biology, and particularly belongs to a polypeptide sequence for screening and identifying a CIM-ScFv antibody based on a murine natural single-chain antibody library and application thereof.
Background
Cimaterol (CIM), which is commonly used by illegal feeding enterprises as a novel clenbuterol instead of kreteron. It has been demonstrated that CIM drug residues in animal food tissues (e.g., lung, liver, kidney, and muscle, etc.) pose a potential risk to consumer health. The use of CIM as a livestock growth promoter has been banned in many countries and regions, but the act of abuse of CIM is still widespread in various regions in order to increase economic profits. In order to assist in solving the problem of food safety and improving the phenomenon that illegal additives are forbidden in China and are forbidden frequently, and continue to perfect a medicine residue monitoring system, the research screens and identifies the polypeptide sequence of a CIM-ScFv antibody based on a murine natural single-chain antibody library, aims to enrich the residue detection method of
Disclosure of Invention
The invention aims to provide a polypeptide sequence for screening and identifying a CIM-ScFv antibody based on a murine natural single-chain antibody library and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
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.
The application of the polypeptide sequence for identifying the CIM-ScFv antibody in the cimaterol antigen identification is screened based on a murine natural single-chain antibody library.
The application of the polypeptide sequence for screening and identifying the CIM-ScFv antibody in the murine natural single-chain antibody library in the rapid detection of the cimaterol antigen.
The application of the polypeptide sequence for identifying the CIM-ScFv antibody in the preparation of the kit for detecting the cimaterol residue in the sample is screened based on the murine natural single chain antibody library.
Further preferably, the sample includes, but is not limited to, animal tissue, urine, and feed.
Further, the kit includes, but is not limited to, an ELISA kit.
The application of the polypeptide sequence for identifying the CIM-ScFv antibody in immune related diagnostic products is screened based on a murine natural single chain antibody library.
More preferably, the immune-related diagnostic product includes, but is not limited to, a WB kit, an ELISA kit, a colloidal gold reagent strip and a fluorescent diagnostic reagent
Compared with the prior art, the invention has the following characteristics and beneficial effects
The sequence shown as SEQ ID NO.1 provided by the invention is as follows: ARWTVITYAMDY, and the sequence shown in SEQ ID NO. 2: QNGHSFPYT, the specificity is better, and the cimaterol antibody can be synthesized quickly according to the sequence, so that the quick artificial synthesis can be realized.
The invention firstly designs a primer to amplify a mouse antibody gene variable region, synthesizes a single-chain antibody thereof, constructs a recombinant phagemid pCANTAB-5E-ScFv, carries out electrotransformation into host bacteria TG1, assists in rescue of phage M13K07, obtains a murine phage single-chain antibody library, identifies, uses a cimaterol complete antigen as a target, carries out four-wheel screening, carries out quality evaluation on the obtained single-chain antibody, shows that the phage recovery rate in the four-wheel screening always increases, shows that specific phage antibodies are effectively enriched along with the increase of the elutriation times, carries out affinity identification with the cimaterol antigen, has better affinity, can be applied to the production of a rapid detection reagent of the cimaterol antigen, enriches a residue detection method of
Drawings
FIG. 1 is a schematic representation of four effective enrichments of specific phage antibodies;
FIG. 2 is a diagram showing the result of mouse RNA electrophoresis;
FIG. 3 is a schematic representation of the electrophoresis results of VH and VL;
FIG. 4 is a schematic representation of the SOE-PCR splicing ScFv antibody gene;
FIG. 5 is a graphic representation of the electrophoresis results of ScFv amplification;
FIG. 6 is a graphical representation of the results of an assay to determine antibody repertoire titers;
FIG. 7 is a schematic representation of SDS-PAGE detection of soluble ScFvs;
FIG. 8 soluble ScFvic-ELISA test results are shown in the figure.
Detailed Description
In order to make the technical means, innovative features, objectives and functions realized by the present invention easy to understand, the present invention is further described below.
The examples described herein are specific embodiments of the present invention, are intended to be illustrative and exemplary in nature, and are not to be construed as limiting the scope of the invention. In addition to the embodiments described herein, those skilled in the art will be able to employ other technical solutions which are obvious based on the disclosure of the claims and the specification of the present application, and these technical solutions include any obvious replacement or modification of the embodiments described herein
The invention firstly extracts total RNA from fresh spleen tissue of non-immunized healthy BABL/c mice, and then amplifies total antibody variable region cDNA by RT-PCR. Using the coding pentapeptide (Gly)4Ser)3The purified products of the VH and VL genes were assembled into a complete mouse ScFv antibody gene. Finally cloning the recombinant ScFv fragment to a phage pCANTAB-5E vector, carrying out electroporation to transfer the recombinant ScFv fragment to Escherichia coli TG1, obtaining a mouse-derived recombinant phage antibody library of a functional single-chain fragment variable region (ScFv) by reinfection of an auxiliary phage M13KO7, identifying the library, carrying out four-wheel screening on the obtained single-chain antibody by taking a cimaterol complete antigen as a target, carrying out quality evaluation on the obtained single-chain antibody, drawing by taking the phage recovery rate of each wheel as a vertical coordinate, as shown in figure 1, showing that the phage recovery rate always increases, showing that specific phage antibodies are effectively enriched along with the increase of the elutriation times, selecting a single colony for amplification culture, sending to a company Limited in the engineering (Shanghai) for sequencing, wherein the sequencing primers are pCANTAB-5 MIGAE universal primers S1 and S6, removing enzyme cutting sites and protecting bases, and predicting by a T database, the sequence shown in SEQ ID NO.1 and the sequence shown in SEQ ID NO.2 of the invention are obtained, the soluble expression and the property identification are carried out on the screened positive phage clone of the 13-CIM-ScFv, the titer of the soluble ScFv antibody generated by the hybridoma cell strain to CIM is about 1:320 through identification, the antibody titer is higher, the antibody subtype is IgG1, the antibody affinity is high,the kit can be used for detecting cimaterol drug residues, and the soluble ScFv antibody specifically reacts with antigen CIM but does not react with BSA and OVA, which indicates that the No. 13 phage (13-CIM-ScFv) antibody has good specificity to CIM.
The following is a detailed description of the experimental contents to which the present invention relates:
1. obtaining a murine recombinant phage antibody library of a single-chain fragment variable region (ScFv);
1.1 extraction and identification of mouse RNA
One healthy, uninmmunized mouse of 6-8 weeks of age was selected and fixed on a clean wax tray with the abdomen facing up. After the abdomen was sterilized with an alcohol cotton ball, two sets of sterilized scissors forceps were prepared, one set was used to cut the skin, and the other set was used to cut the peritoneum and then the spleen was removed. Removing adhered tissue with scissors, quickly transferring intact spleen into a mortar (sterilized by autoclaving) pre-cooled at-80 deg.C, and adding small amount of liquid nitrogen during grinding to make grinding more complete. This process takes care of low temperatures, prevents RNA degradation, and destroys gene integrity. The spleen tissues which are fully cracked are used for extracting RNA according to the operation steps of the kit.
Diluting the extracted RNA by 10 times with TE buffer solution or RNAase-free Water, preliminarily identifying the diluent with a trace protein nucleic acid analyzer, repeatedly correcting for 3 times with the TE buffer solution or RNAase-free Water as a blank sample, wiping the contact for detection, and recording the ratio of A260/A280 and the concentration of nucleic acid; and finally, mixing 5 mu L of RNA diluent with 1 mu L of 6 × Loading buffer, Loading the mixture, carrying out voltage 150 v, and viewing the result by ultraviolet imaging of a gel imager after 15 min. When the complete RNA is subjected to agarose electrophoresis, two bands of 28S and 18S rRNA appear, the brightness of the former band is about one time stronger than that of the latter band, and the specific result is shown in figure 2, so that the extracted RNA has good integrity and can be used for subsequent PCR amplification reaction.
1.2 Synthesis of first Strand of mouse cDNA
Preparing RT reaction liquid from the identified complete RNA on ice according to the specification of a reverse transcription kit, mixing the reaction liquid uniformly, setting PCR reaction in the PCR, and directly using the cDNA after the reaction for the amplification of a target fragment or storing the cDNA at-20 ℃ for later use.
1.3 primer design and Synthesis
The mouse antibody gene is downloaded by NCBI and IMGT databases, the nucleic acid sequence of the mouse source antibody variable region is analyzed by DNAMAN software in a comparison way, and specific primers are designed by using Primer 5.0 software for amplifying the BABL/c mouse heavy chain variable region (VH) and light chain variable region (VL) genes.
1.4 Synthesis of VH and VL
Using the first chain of cDNA as template, PCR amplifying to synthesize VH and VL of mouse gene, preparing PCR reaction system, using mixed primers as VH and VL primers for variable region amplification, calculating the relative proportion of each primer in the mixed primers according to the degeneracy degree of the primers, so as to make the usage of each unique primer represented by degenerate basic group equal. And (3) carrying out PCR amplification reaction after the reaction solution is uniformly mixed in a vortex mode and centrifuged for a short time, carrying out electrophoresis identification on PCR amplification products, wherein the result is shown in figure 3, then recovering the PCR amplification products, storing the recovered products at the temperature of-20 ℃, wherein the size of the VH fragment is 432 bp (figure 3A), the size of the VL fragment is 312 bp (figure 3B), and the method accords with the expected result and can be used for ScFv synthesis.
As shown in Table 1, since there is a complementary overlap region of 30bp between the VH forward primer and the VL forward primer, the above-mentioned VH and VL collected products were ligated without primers (shown in FIG. 4 as SOE-P1) by the overlap extension PCR method (SOE-PCR), and assembled into an ScFv gene product, and then a large amount of the ScFv antibody gene fragment was amplified by adding a primer (shown in FIG. 4 as SOE-P2). Therefore, after the VH and VL are amplified in a large quantity, the VH and VL are recovered and purified by a gel recovery kit, an SOE-PCR method is adopted to synthesize the ScFv full-length gene, the result is shown in figure 5, the fragment size is about 780 bp, after electrophoretic identification, the VH and VL are amplified in a large quantity and recovered, and the recovered product is the mouse single-chain antibody gene. Preparing PCR reaction solution from a sterilized PCR micro reaction tube, and storing PCR amplification products at-20 ℃ for identification and construction of recombinant vectors.
1.5 construction of phage antibody libraries
1.5.1 preparation of recombinant vector pCANTAB-5E-ScFv
The purified ScFv gene was ligated with vector pCANTAB-5E to construct a recombinant phage vector. And (3) configuring a connecting system, uniformly mixing, and connecting in a low-temperature water bath kettle at 16 ℃ for 10-12 h. The ligation product was added to TG1 electroporation competent cells and the mixture was immediately transferred to a clean, dry, pre-chilled cuvette for electroporation.
Immediately adding 1 mL of SOC culture medium (preheated at 37 ℃ and nutrient content higher than that of a common culture medium) into the click cup after electric shock is finished, slightly blowing and beating the click cup, transferring the click cup into a new bacteria shaking tube, and carrying out shake culture at 37 ℃ and 200 rpm for 1 h to rejuvenate the strains; centrifuging at 5000 rpm for 5 min, retaining about 200 μ L of supernatant, discarding the rest supernatant, re-suspending the cells, spreading on 2 XYT-A solid medium (containing ampicillin 100 μ g/mL), and culturing in 37 deg.C incubator overnight.
The n mutext day, single colony with clear edge is picked from the plate, cultured in 5 mL2 × YT-A liquid culture medium at 37 ℃ and 180 rpm overnight, 1 mL of the bacterial liquid after shake culture is taken, centrifuged, supernatant is discarded, 20 mu L of sterile water is used for resuspending the thallus and is used as a template for reaction for PCR identificationSfiI、ScFvRevNot1The reaction system and the reaction procedure are the same as those of SOE-PCR.
1.6 determination of antibody library titres
(1) The rescued phage antibody library was diluted with 2 × YT medium (or PBS buffer) and the procedure was as follows: adding 1 mu L of antibody library stock solution (diluted by 1: 100) into 99 mu L of 2 XYT culture solution, and uniformly mixing. The method is characterized in that 10 muL of the mixed solution is taken to be placed in 90 muL of 2 XYT culture solution, namely the phage solution diluted by 1000 times, so that the mixed solution is diluted by multiple orders of magnitude, and proper and accurate dilution is the key point for the good gradient phenomenon.
(2) From order of magnitude 102、104、106、108Taking out 10 muL of the dilution, respectively adding the dilution into 90 muL of TG1 culture solution (OD 600= 0.4), co-culturing for 30 min at 37 ℃ (the same as the measurement of the titer of M13K 07), uniformly mixing the dilution with 3 mL of top agar, pouring the mixture into a2 × YT-KA plate, calculating the number of plaques on the plate the next day100 μ L of culture solution TG1 in logarithmic phase of the somatic library was cultured in the same manner. The colony number on the plate is reduced along with the increase of the dilution multiple of the phage library, the TG1 plate without the added phage has no single colony, the result is reliable, and if the colony is not added, the plaque needs to be picked again to repeat the construction and rescue of the phage.
The growth of the dilutions of the antibody library of this experiment on the plates is shown in FIG. 6. Antibody library channels 108After dilution, the number of effective single colonies grown on the plate was 156, and the titer of the phage ScFv antibody library obtained in this study was 1.56 × 10 according to the titer calculation formula10pfu。
Soluble expression and property identification of 2-anti-cimaterol single-chain antibody in escherichia coli
Soluble expression of 2.113-CIM-ScFv
Single colonies with good isolation and clear edges were picked, inoculated into 5 mL2 XYT-A medium, shake-cultured overnight at 30 ℃ and 250 rpm, and 500. mu.L overnight culture was taken the n mut mutext day, shake-cultured to logarithmic phase at 30 ℃ and 250 rpm in 50 mL2 XYT-A medium. Adding IPTG to a final concentration of 0.25, 0.1 and 0.5 mmol/L, culturing at 30 ℃, adopting different induction conditions and different induction time, centrifuging after induction, 10000 rpm, collecting precipitates after 20 min, fully resuspending with 2 mL of precooled TBS, standing at-20 ℃ for 20 min, standing at room temperature for 20 min, 10000 rpm and 20 min, transferring the supernatant to a new tube for later use, resuspending the bacteria with 1 mL of hypertonic extracting solution (precooling on ice), vortexing and shaking at room temperature for 10 min, standing at 4 ℃ for 10 min, centrifuging at low temperature for 10000 rpm, 20 min, discarding the supernatant, adding isovolume of precooled hypotonic buffer solution, gently stirring at low temperature for 30 min, centrifuging at 4 ℃, 10000 rpm and 20 min, and reserving the supernatant, wherein the supernatant contains ScFv protein secreted into periplasm.
2.2 SDS-PAGE detection of soluble ScFv
After the samples were treated, SDS-PAGE was performed on the phage-positive ScFv proteins under different induction conditions and the culture supernatants of the control bacteria, respectively. The blank control had no band, and the supernatant of the uninduced bacteria solution had more impurity. Protein bands appear in the induced bacterial liquid at about 30KDa, the sizes are expected, the bands are single, and excessive protein is avoided. Indicating that soluble expression of ScFv antibody against cimaterol is present in the bacterial periplasm of E.coli HB 2151. As shown in FIG. 7, the optimal induction conditions were 0.1mmol/L IPTG, 30 ℃ and 16 hours. The induction conditions were selected as induction conditions for the large-scale preparation of 13-CIM-ScFv. The results indicate that the protein is not frameshifted during expression.
2.3 preliminary establishment of competitive inhibition ELISA assay for soluble ScFv
2.3.1 working concentration determination of purified antibodies
The titer of ScFv protein secreted into the periplasm of cells was determined by indirect ELISA. Note: the periplasmic cavity extraction antibody was diluted 1:5 in primary antibody dilution. The secondary antibody was Anti-E-tag (HRP) antibody 1:4000 dilution, and the negative control was pCANTAB-5E-HB 2151. The absorbance value is about 1.0, and the P/N ratio is more than 2.1, which corresponds to the optimal concentration of ScFv protein for competitive inhibition ELISA.
The results show that the antibody can react with the coating antigen. Indicating that the single chain antibody was correctly folded and expressed. As shown in table 1, greater than the OD of the negative well450The antibody titer to CIM was determined to be about 1:320 by determining the maximum dilution factor corresponding to the positive well as 2.1 times of the reading as the titer of the antibody.
Table 1 results of measuring soluble ScFv titer by indirect ELISA.
Dilution factor (Serum dilution)
1:5
1:10
1:20
1:40
1:80
1:160
1:320
1:640
Negative control (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 competitive inhibition ELISA Standard Curve and determination of sensitivity and minimum detection Limit
8 standard concentrations are selected to establish a standard curve, and the 50% inhibition concentration of the added standard substance to the envelope antigen is taken as the median inhibition concentration (IC 50), wherein the lower the IC50 value is, the lower the inhibition effect is, the higher the sensitivity is. The determination method of the lowest detection line is the concentration of the corresponding small molecular 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 substituting the standard curve.
Specifically, the soluble antibody was selected as the primary antibody after dilution by a factor of 1: 10. The standard curve established by the logarithm of the 6 standard concentrations (1, 3, 9, 27, 81, 243 ng/mL) and their corresponding absorbance values was y = -0.2561x +1.0358, degree of fitting R2= 0.9848, as in fig. 8. Half inhibition concentration IC50=101 ng/mL, lowest limit of detection LOD =3.16 ng/mL, and good fitting degree is achieved in the range of 1-243 ng/mL in the competitive ELISA method established by the phage antibody.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
SEQUENCE LISTING
<110> college of New county
<120> polypeptide sequence for screening and identifying CIM-ScFv antibody based on murine natural single-chain antibody library and application thereof
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<170>PatentIn version 3.3
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Ala Arg Trp Thr Val Ile Thr Tyr Ala Met Asp Tyr
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Gln Asn Gly His Ser Phe Pro Tyr Thr
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