CN110760483B

CN110760483B - Preparation and application of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction

Info

Publication number: CN110760483B
Application number: CN201911088251.9A
Authority: CN
Inventors: 焦新安; 陈祥; 李昕; 谈悦; 徐正中; 朱兆成; 夏爱鸿; 王蕾; 顾丹
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2021-06-22
Anticipated expiration: 2039-11-08
Also published as: CN110760483A

Abstract

The invention relates to the field of biotechnology, in particular to a hybridoma cell strain, and further relates to an anti-TNF-alpha antibody prepared from the hybridoma cell strain, and a preparation method and application thereof. The invention provides a hybridoma cell strain or a subculture cell strain thereof, wherein the preservation number of the hybridoma cell strain is CCTCC NO: c2019129 and further provides an anti-TNF-alpha antibody produced by the hybridoma cell line or a passaged cell line thereof. The monoclonal antibody provided by the invention has the advantages of high titer and strong binding affinity with natural antigen, and can be used for detecting natural bovine TNF-alpha and sheep TNF-alpha.

Description

Preparation and application of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction

Technical Field

The invention relates to the field of biotechnology, in particular to a hybridoma cell strain, and further relates to an anti-TNF-alpha antibody prepared from the hybridoma cell strain, and a preparation method and application thereof.

Background

After being activated, the immune system of the body produces a cytokine which can cause hemorrhagic necrosis of tumors and is named as tumor necrosis factor alpha (TNF-alpha). TNF- α is a cytokine involved in systemic inflammation and is secreted primarily by macrophages. Its main function is to regulate immune cells, as an endogenous pyrogen, can cause fever, induce apoptosis, prevent tumorigenesis and virus replication, etc. Changes in TNF- α expression levels have certain relevance to the development of infectious diseases, immune dysfunction, inflammatory diseases, tumors, and the like.

The immunological detection of TNF-alpha is an experimental method for qualitative and quantitative analysis of TNF-alpha in a sample, which is established on the basis of a specific anti-TNF-alpha monoclonal antibody (MAb). By applying different monoclonal antibody markers and detection technologies, various detection methods can be established, such as Enzyme-linked Immunosorbent Assay (ELISA), Flow Cytometry (FCM) analysis and the like. As one of important indexes of the cellular immune state, immunological detection methods of TNF- α are widely used in basic research and clinical medicine, vaccine immune effect evaluation, disease diagnosis, allergic reaction, and the like.

However, the anti-bovine and ovine TNF- α antibodies prepared in the prior art often suffer from great problems when applied to the above detection methods, and particularly have poor effects when used for detecting natural bovine and ovine TNF- α.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a hybridoma cell line, and further provides an anti-TNF- α antibody prepared from the hybridoma cell line, and a preparation method and use thereof, which are used for solving the problems in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a hybridoma cell strain or a passaged cell strain thereof, wherein the hybridoma cell strain has a preservation number of CCTCC NO: C2019129.

the invention also provides an anti-TNF-alpha antibody, which is prepared from the following components in percentage by weight: c2019129 or a subculture cell strain thereof.

In another aspect, the present invention provides an anti-TNF- α antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the CDR of the light chain variable region comprises CDR-L1 having an amino acid sequence as set forth in SEQ ID No.1, CDR-L2 having an amino acid sequence as set forth in SEQ ID No.2, and CDR-L3 having an amino acid sequence as set forth in SEQ ID No.3, and the CDR of the heavy chain variable region comprises CDR-H1 having an amino acid sequence as set forth in SEQ ID No.6, CDR-H2 having an amino acid sequence as set forth in SEQ ID No.7, and CDR-H3 having an amino acid sequence as set forth in SEQ ID No. 8.

In some embodiments of the invention, the anti-TNF- α antibody is a monoclonal antibody.

In some embodiments of the invention, the amino acid sequence of the light chain variable region of the anti-TNF- α antibody comprises:

a) an amino acid sequence shown as SEQ ID No. 4; or

b) An amino acid sequence which has more than 80 percent of homology with the amino acid sequence shown in SEQ ID No.4 and has the amino acid sequence function defined by a).

In some embodiments of the invention, the amino acid sequence of the light chain of the anti-TNF- α antibody includes the amino acid sequence set forth as SEQ ID No. 5.

In some embodiments of the invention, the amino acid sequence of the heavy chain variable region of the anti-TNF- α antibody comprises:

c) an amino acid sequence shown as SEQ ID No. 9; or

d) An amino acid sequence which has more than 80 percent of homology with the amino acid sequence shown in SEQ ID No. 9and has the amino acid sequence function defined by c).

In some embodiments of the invention, the amino acid sequence of the heavy chain of the anti-TNF- α antibody includes the amino acid sequence set forth as SEQ ID No. 10.

In another aspect, the invention provides an isolated polynucleotide encoding a heavy chain variable region and/or a light chain variable region and/or a full length amino acid of said anti-TNF- α antibody.

In another aspect, the invention provides a construct comprising the isolated polynucleotide.

In another aspect, the invention provides an antibody expression system comprising the construct or the polynucleotide integrated into the genome from an exogenous source.

In another aspect, the present invention provides a method for preparing the anti-TNF- α antibody, comprising the steps of: culturing said antibody expression system to express said antibody, and purifying and isolating said antibody;

and/or the expression vector is expressed by a collection number of CCTCC NO: hybridoma production of C2019129.

The invention also provides application of the anti-TNF-alpha antibody in preparing a TNF-alpha detection kit.

In another aspect, the invention provides a detection kit comprising the anti-TNF-alpha antibody.

Drawings

FIG. 1 is a graph showing the detection results of TNF- α proteins in cattle and sheep by the Western-Blot method of example 5 of the present invention: Western-Blot detection results of rHis-BoTNF-alpha and rGST-BoTNF-alpha proteins; Western-Blot detection results of rHis-ShTNF-alpha and rGST-ShTNF-alpha proteins.

FIG. 2 shows the direct immunofluorescence (DFA) assay results of bovine and ovine peripheral blood mononuclear cell samples in example 9 of the present invention. A, detecting results of PMA and ionomycin combined stimulation of bovine peripheral blood mononuclear cells; B. detecting results of unstimulated bovine peripheral blood mononuclear cells; PMA and ionomycin are combined to stimulate the detection result of the sheep peripheral blood mononuclear cells; d, detecting the unintimulated sheep peripheral blood mononuclear cells.

FIG. 3 is a graph showing the result of detecting bovine and ovine peripheral blood mononuclear cell samples by the FCM kit in the experiment of example 11 of the present invention: A. detecting results of non-stimulated bovine peripheral blood mononuclear cells and PMA combined with ionomycin stimulated bovine peripheral blood mononuclear cells; B. and (3) detecting the result of the sheep peripheral blood mononuclear cell not stimulated and the PMA combined with the ionomycin stimulated sheep peripheral blood mononuclear cell.

FIG. 4 shows the result of detecting bovine peripheral blood mononuclear cell sample by the FCM detection kit for bovine tuberculosis in the experiment of example 13 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

The inventor of the invention provides a hybridoma cell strain through a great deal of research, and further relates to an anti-TNF-alpha antibody prepared by the hybridoma cell strain, wherein the anti-TNF-alpha antibody can be combined with natural bovine and ovine TNF-alpha with high affinity, and has high sensitivity and high specificity, and the invention is completed on the basis.

The first aspect of the invention provides a hybridoma cell strain or a subculture cell strain thereof, wherein the preservation number of the hybridoma cell strain is CCTCC NO: c2019129, which is preserved in China center for type culture Collection, wherein the preservation address is China, Wuhan university, and the preservation date is 2019, 6 months and 17 days.

The second aspect of the invention provides an anti-TNF-alpha antibody, which is prepared from the following components in percentage by weight with the preservation number of CCTCC NO: c2019129 or a subculture cell strain thereof. The anti-TNF-alpha antibody provided by the invention can be obtained by secreting the hybridoma cell strain or the subculture cell strain thereof provided by the first aspect of the invention. The anti-TNF-alpha antibody is typically a monoclonal antibody. A monoclonal antibody generally refers to a population of antibodies in which the antibodies included are substantially identical (except for a few naturally occurring mutations that may be present). Monoclonal antibodies are typically directed against a specific determinant on an antigen. The anti-TNF- α antibody may be murine. The anti-TNF-alpha antibody can be combined with natural bovine and ovine TNF-alpha with high affinity, only reacts with rBoTNF-alpha and ShTNF-alpha (natural TNF-alpha of bovine and ovine) in the specificity identification of the antibody, but not reacts with other prokaryotic expression recombinant cytokines, has extremely high titer in titer determination, and has the characteristics of high sensitivity and high specificity.

In a third aspect, the present invention provides an anti-TNF- α antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the CDR of the light chain variable region comprises CDR-L1 having an amino acid sequence as set forth in SEQ ID No.1, CDR-L2 having an amino acid sequence as set forth in SEQ ID No.2, and CDR-L3 having an amino acid sequence as set forth in SEQ ID No.3, and the CDR of the heavy chain variable region comprises CDR-H1 having an amino acid sequence as set forth in SEQ ID No.6, CDR-H2 having an amino acid sequence as set forth in SEQ ID No.7, and CDR-H3 having an amino acid sequence as set forth in SEQ ID No. 8.

A CDR (complementary determining region) generally refers to a region of an antibody that can sterically complement an antigenic determinant. The variability in antibodies is typically not evenly distributed throughout the variable region of the antibody, and the heavy and light chain variable regions of a monoclonal antibody typically each have 3 hypervariable regions (HVRs) which are generally complementary in spatial structure to antigenic determinants, so the hypervariable regions are also referred to as Complementarity Determining Regions (CDRs), i.e., the heavy chain variable region typically includes three complementarity determining regions, CDR-H1, CDR-H2 and CDR-H3, and the light chain variable region typically includes three complementarity determining regions, CDR-L1, CDR-L2 and CDR-L3.

In the anti-TNF- α antibody, the amino acid sequence of the light chain variable region of the anti-TNF- α antibody may include:

a) an amino acid sequence shown as SEQ ID No. 4; or

b) An amino acid sequence which has more than 80 percent of homology with the amino acid sequence shown in SEQ ID No.4 and has the amino acid sequence function defined by a). Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID No.4 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminal and/or C-terminal, and has the amino acid sequence shown in SEQ ID No. 4. The amino acid sequence in b) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% homology with SEQ ID No. 4.

In a specific embodiment of the invention, the amino acid sequence of the light chain of the anti-TNF- α antibody may comprise the amino acid sequence shown as SEQ ID No. 5.

In the anti-TNF- α antibody, the amino acid sequence of the heavy chain variable region of the anti-TNF- α antibody comprises:

c) an amino acid sequence shown as SEQ ID No. 9; or

d) An amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID No. 9and having the amino acid sequence function defined in c); specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown as SEQ ID No.9 is obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids at the N-terminal and/or C-terminal, and has the amino acid sequence shown as SEQ ID No. 9. The amino acid sequence in d) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% homology with SEQ ID No. 9.

In a specific embodiment of the invention, the amino acid sequence of the heavy chain of the anti-TNF- α antibody may comprise the amino acid sequence shown as SEQ ID No. 10.

In a fourth aspect, the invention provides an isolated polynucleotide encoding the heavy chain variable region and/or the light chain variable region and/or the full length amino acids of an anti-TNF- α antibody as provided in the third aspect of the invention.

In a fifth aspect, the invention provides a construct comprising a polynucleotide as provided in the fourth aspect of the invention. The construct may be constructed by inserting the isolated polynucleotide into a multiple cloning site of an expression vector. The expression vector of the present invention is generally referred to various commercially available expression vectors well known in the art, and may be, for example, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.

In a sixth aspect, the present invention provides an antibody expression system comprising a construct or genome provided by the fifth aspect of the present invention and an exogenous polynucleotide provided by the fourth aspect of the present invention integrated therein. Any cell suitable for expression of an expression vector may be used as the host cell, for example, the host cell may be a prokaryotic cell, such as a bacterial cell or the like; or lower eukaryotic cells such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

The seventh aspect of the present invention provides a method for producing the anti-TNF- α antibody provided by the second aspect of the present invention, or the third aspect, which may comprise the steps of: culturing the antibody expression system of claim 11 under conditions suitable for expression of said antibody, thereby expressing said antibody, and purifying and isolating said antibody. Suitable conditions for expression of the antibody will be known to those skilled in the art and one skilled in the art can empirically select a suitable medium for culturing under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The anti-TNF-alpha antibody can also be prepared from a polypeptide with a preservation number of CCTCC NO: the hybridoma production of C2019129, the preparation method may comprise the steps of: is prepared by adopting an in-vivo induced ascites method. Suitable methods for inducing ascites using hybridomas in vivo to provide monoclonal antibodies will be known to those skilled in the art, who can empirically inoculate mice with hybridoma cells and collect ascites. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

In an eighth aspect, the invention provides the use of an anti-TNF α antibody provided by the second or third aspects of the invention in the preparation of a TNF- α detection kit.

In an eighth aspect, the invention provides a test kit comprising the anti-TNF- α antibody of the second or third aspects, particularly in a diagnostically effective amount, which generally refers to an amount that provides a diagnostic benefit. Therefore, the diagnostic kit can generally aim at the TNF-alpha antigen serving as a target and perform diagnosis by taking the TNF-alpha antigen as a biomarker. The diagnostic kit may also include a label for an anti-TNF-alpha antibody that may be used to label the anti-TNF-alpha antibody generally, and the types of labels that may be selected include, but are not limited to, combinations of one or more of fluorescent labels, radioactive labels, enzyme labels, chemiluminescent labels, and the like. Depending on the detection principle of the kit, the kit may also typically comprise one or more reagents required for the detection. In addition, the kit can also comprise the following components according to needs: containers, controls (negative or positive controls), buffers, adjuvants, etc., which can be selected by one skilled in the art as appropriate.

In a specific embodiment of the invention, the kit can be a Western-Blot detection kit, and can be used for detecting natural bovine or ovine TNF-alpha protein secreted by bovine or ovine peripheral blood mononuclear cells. The Western-Blot detection kit can comprise the labeled anti-TNF-alpha antibody, specifically can be the anti-TNF-alpha antibody labeled by horseradish peroxidase, and can also comprise other reagents, for example, the reagents can be one or a combination of more of confining liquid, substrate liquid, washing liquid and the like.

In another embodiment of the invention, the kit can be a direct immunofluorescence (DFA) detection kit, and the detection kit is used for a method for detecting TNF-alpha protein of cattle and sheep taking Hela cells as expression hosts, and can also be used for analyzing cattle and sheep peripheral blood mononuclear cell samples. The direct immunofluorescence (DFA) detection kit may include the anti-TNF- α antibody labeled, specifically fluorescein isothiocyanate labeled, and may include other reagents, e.g., may be a combination of one or more of a fixative, a washing solution, and the like.

In another embodiment of the present invention, the kit may be an FCM detection kit, and the kit may be used for detecting monocytes secreting TNF- α in cattle and sheep, thereby performing studies on the evaluation of the immune status of the body and the diagnosis of diseases. The FCM detection kit may include the labeled anti-TNF- α antibody, specifically, the fluorescein isothiocyanate labeled anti-TNF- α antibody, and the direct immunofluorescence detection kit may include other reagents, for example, one or a combination of more of a blocking agent, a fixing agent, a membrane breaking agent, a washing solution, and the like.

A ninth aspect of the present invention provides a detection method, including: the bovine and/or ovine TNF- α in a sample is detected by the anti-TNF- α antibody provided by the second or third aspects of the invention, or the detection kit provided by the eighth aspect of the invention. The detection can be for non-diagnostic treatment purposes, and the non-diagnostic detection comprises the detection of recombinant expression of bovine and ovine TNF-alpha, the detection of isolated tissues and epitope identification research.

The monoclonal antibody provided by the invention has the advantages of high titer and strong binding affinity with natural antigen, and can be used for detecting natural bovine TNF-alpha and sheep TNF-alpha. The Western-Blot detection kit established based on the monoclonal antibody provided by the invention can effectively detect the TNF-alpha proteins of cattle and sheep expressed by an escherichia coli expression system, and can also detect the natural TNF-alpha proteins of cattle and sheep secreted by Peripheral Blood Mononuclear Cells (PBMC) of cattle and sheep; the direct immunofluorescence (DFA) detection kit established based on the monoclonal antibody provided by the invention can effectively detect bovine and ovine TNF-alpha expressed and secreted by bovine and ovine peripheral blood mononuclear cell samples and eukaryotic plasmids; the FCM detection kit for the TNF-alpha of the cattle and the sheep, which is established based on the monoclonal antibody provided by the invention, can detect the peripheral blood mononuclear cells of the cattle and the sheep which secrete the TNF-alpha of the cattle and the sheep at a higher level when detecting the peripheral blood mononuclear cell samples of the cattle and the sheep. Therefore, the monoclonal antibody and the related kit thereof provided by the invention are simple and convenient to operate, greatly shorten the detection time, and can be widely applied to immunological research, the research related to the evaluation of the immune state of a cattle body and the diagnosis of infectious diseases, and the detection of the TNF-alpha of cattle and sheep with non-diagnosis purposes.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1: obtaining of hybridoma cell lines

The hybridoma cell strain with the preservation number of CCTCC NO of C2019129 is obtained.

1. Purification of rHis-BoTNF-alpha recombinant protein

Designing a primer according to a BoTNF-alpha cDNA sequence (XM-005223596.4) in GenBank, and expressing a primer sequence P1: 5' -ACAAGGCCATGGCTGATATC of a recombinant vector pET-30a (+) -BoTNF-alphaGGATCCCTCAGGTCCTCTTCTCAA-3 '(the underlined part is the BamHI cleavage site) P2: 5' -TGGTGCTCGAGTGCGGCCGCAAGCTTTCACAGGGCGATGATCCCAAAGTA-3' (underlined is the Hind III site).

Total bovine peripheral blood lymphocyte RNA was extracted and reverse transcribed according to the RNeasy plus Mini kit instructions. The amplification of the BoTNF-alpha gene and the double digestion of the pET-30a (+) vector. Mixing the recovered and purified vector and the target gene at a molar mass ratio of 1:3, adding one-step method ligase, reacting at 37 ℃ for 30min, connecting the target gene and the vector, and performing heat transfer to BL21(DE 3). The recombinant strain was named BL21(DE3) (pET-30a (+) -BoTNF-. alpha.). Then recombinant bacteria BL21(DE3) (pET-30a (+) -BoTNF-alpha) are induced, expressed and purified to obtain rHis-BoTNF-alpha purified protein.

2. Animal immunization

The specific immunization program was as follows: the BALB/c mouse is immunized for the first time, 100 mu g of rHis-BoTNF-alpha purified protein fully emulsified by Freund's complete adjuvant is injected into the abdomen at multiple points, after 2 weeks, 100 mu g of purified protein fully emulsified by Freund's incomplete adjuvant is injected into the abdomen at multiple points for secondary immunization, after 2 weeks, 100 mu g of purified protein without adjuvant is injected into the abdomen for third immunization, blood is collected after 7 days to determine the titer of serum antibody, and the mouse with higher titer is selected to perform intraperitoneal boosting immunization on 100 mu g of purified protein without adjuvant.

3. Cell fusion

The method comprises the following specific steps: after 3 days of intraperitoneal injection for boosting, a small amount of blood is collected, serum is separated, and the blood is frozen and stored at the temperature of minus 20 ℃ to be used as a positive clone control in screening. Aseptically taking spleen cells of immunized mice and myeloma cells SP2/0 in logarithmic growth phase according to a biosafety method, fusing under the action of polyethylene glycol PEG (MW1500), taking ICR mouse abdominal cavity macrophages as feeder cells, suspending the fused cells and feeder cells by HAT culture medium, subpackaging with 96-well plates, placing at 37 ℃, and placing with 5% CO₂Culturing in an incubator. Adding fresh HAT culture medium after 5 days, culturing with HT culture medium after 10 days, periodically observing, changing liquid and detecting.

4. Establishment of indirect ELISA detection method

Screening positive clone cells by adopting an indirect ELISA method. The matrix test determines the coating concentration of the test antigen.

The detection antigen is diluted by a coating buffer solution in a transverse gradient way, each hole is coated with 100 mu L of an ELISA plate, and the temperature is kept overnight at 4 ℃; PBST is washed for 3 times, 200 mu L of blocking solution is added into each hole, and incubation is carried out for 2h at 37 ℃; diluting immune mouse serum longitudinally by multiple, wherein each hole is 100 mu L, diluting normal mouse serum by the same multiple as negative control, and incubating at 37 ℃ for 2 h; washing with PBST for 3 times, adding enzyme-labeled secondary antibody with working concentration, incubating at 37 deg.C for 1h, wherein each well is 100 μ L; after PBST washing, 3',5,5' -Tetramethylbenzidine (TMB) is added for color development, and OD is measured by an enzyme-linked detector₄₅₀The optimal coating concentration of the detection antigen is determined.

According to the coating concentration of the detection antigen determined by the square matrix test, 100 mu L/hole of the diluted detection antigen is added into an enzyme label plate, the temperature is 4 ℃ overnight, PBST is washed for 3 times and 5 min/time, PBST containing 1% BSA (bovine serum albumin) is used for sealing at 4 ℃ overnight, and after PBST is washed, the plate is dried and stored at the temperature of-20 ℃ for screening positive clone cells.

5. Screening for Positive clones

And detecting the condition that the hybridoma cells secrete the antibody by adopting an established indirect ELISA method. The specific method comprises the following steps: subjecting the hybridoma to cell proliferationAdding the cell supernatant into an ELISA plate coated with the optimal coating concentration determined in the step 3 in advance, taking 100 mu L/hole, taking SP2/0 cell supernatant as a negative control, taking immune mouse polyclonal antiserum as a positive control, and carrying out water bath at 37 ℃ for 2 h; PBST wash 3 times; adding horse radish peroxidase HRP-labeled goat anti-mouse IgG with working concentration, 100 mu L/hole, and performing water bath at 37 ℃ for 1.5 h; washing, adding TMB for developing for 10-15min, and measuring OD with enzyme-labeling instrument after the development is terminated₄₅₀And (6) reading. Measured hole OD₄₅₀The reading is more than 2 times larger than the negative control, and the result is judged to be positive. The positive clone selected was named 3C 1.

6. Cloning of Positive hybridoma cells

The selected positive cell clone 3C1 was subcloned 3 times by limiting dilution and stored. The positive cell clone 3C1 corresponds to a hybridoma cell strain with the preservation number of CCTCC NO: C2019129.

Example 2: preparation of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction

1. Preparation of ascites

Adopts a method of inducing ascites in vivo and is carried out according to a conventional method. Injecting liquid paraffin 0.3-0.5 mL/mouse into abdominal cavity of 10-12 week-old healthy BALB/C mouse, inoculating hybridoma cells 3C1, 5 × 10 diluted with PBS and cultured to logarithmic phase growth into abdominal cavity after 7-10 days⁵One cell/one; seven days later, ascites was collected, the precipitate was removed by centrifugation, and the supernatant was collected and stored at-70 ℃.

2. Antibody purification

The prepared monoclonal antibody mAb3C1 ascites fluid was purified using Protein A affinity chromatography.

Example 3: detection of monoclonal antibody characteristics

1. Identification of monoclonal antibody subclasses

According to the specification of the monoclonal antibody subclass kit, an antigen-mediated ELISA method is adopted. Respectively adding 100 mu L/hole of cell culture supernatant into the coated enzyme label plate, washing for 3 times (5 min each time) by PBST at 37 ℃ for 1 h; adding 1:1000 diluted goat anti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3, IgM subclass antibody 50 μ L/well, 0.5h at 37 deg.C, adding monoclonal antibody mAb3C1, and washing with PBST for 3 times, each for 5 min; adding 1:50The rabbit anti-sheep enzyme labeled secondary antibody diluted by 00 is 50 mu L/hole, is washed for 3 times at 37 ℃ for 15min by PBST; adding TMB developing solution 100 μ L/well, developing at 37 deg.C in dark for 10-15min, 2M H₂SO₄The reaction was stopped at 50. mu.L/well, and the antibody subclass was determined to be a monoclonal antibody subclass which was visibly higher in color than the subclass added to the other wells.

The results show that mAb3C1 subclass is IgG 1.

The identification result shows that the amino acid sequence of the complementarity determining region 1(CDR1) of the light chain variable region of the monoclonal antibody 3C1 is shown in SEQ ID NO.1, and specifically comprises the following steps:

RASENIYIYLA(SEQ ID NO.1)

the amino acid sequence of the complementarity determining region 2(CDR2) of the light chain variable region of monoclonal antibody 3C1 is shown in SEQ ID NO.2, and specifically comprises:

NAKTLAE(SEQ ID NO.2)

the amino acid sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 3C1 is shown in SEQ ID NO.3, and specifically comprises:

QHHYGTPWT(SEQ ID NO.3)

the amino acid sequence of the light chain variable region of the monoclonal antibody 3C1 is shown in SEQ ID NO.4, and specifically comprises the following steps:

DIQMTQSPASLSASVGETVTITCRASENIYIYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLNINSLQPEDFGSYYCQHHYGTPWTFGGGTKLEIK(SEQ ID NO.4)

the amino acid sequence of the light chain of the monoclonal antibody 3C1 is shown as SEQ ID NO.5, and specifically comprises the following steps:

MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYIYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLNINSLQPEDFGSYYCQHHYGTPWTFGGGTKLEIK (SEQ ID NO.5), i.e., the light chain of monoclonal antibody 3C1, contains 127 amino acids.

The amino acid sequence of the heavy chain variable region complementarity determining region 1(CDR1) of monoclonal antibody 3C1 is shown in SEQ ID NO.6, and specifically comprises:

NFSVH(SEQ ID NO.6)

the amino acid sequence of the heavy chain variable region complementarity determining region 2(CDR2) of monoclonal antibody 3C1 is shown in SEQ ID NO.7, and specifically comprises:

VMWSGGSTDYNAAFIS(SEQ ID NO.7)

the amino acid sequence of the heavy chain variable region complementarity determining region 3(CDR3) of monoclonal antibody 3C1 is shown in SEQ ID NO.8, and specifically comprises:

SGPYYYSLDY(SEQ ID NO.8)

the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C1 is shown as SEQ ID NO.9, and specifically comprises the following steps:

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNFSVHWVRQPPGKGLEWLGVMWSGGSTDYNAAFISRLSIIKDNSKSQVFFKMNGLQADDTAIYYCARSGPYYYSLDYWGQGTSVTVSS(SEQ ID NO.9)

the amino acid sequence of the heavy chain of the monoclonal antibody 3C1 is shown as SEQ ID NO.10, and specifically comprises the following steps:

MAVLVLLFCLVTFPSCVLSQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNFSVHWVRQPPGKGLEWLGVMWSGGSTDYNAAFISRLSIIKDNSKSQVFFKMNGLQADDTAIYYCARSGPYYYSLDYWGQGTSVTVSS (SEQ ID NO.10), i.e., monoclonal antibody 3C1, has a heavy chain of 137 amino acids.

Correspondingly, the nucleotide sequence of the complementarity determining region 1(CDR1) of the light chain variable region of monoclonal antibody 3C1 is shown in SEQ ID NO.11, specifically:

CGAGCAAGTGAGAATATTTACATTTATTTAGCA(SEQ ID NO.11)

the nucleotide sequence of the complementarity determining region 2(CDR2) in the light chain variable region of monoclonal antibody 3C1 is shown in SEQ ID NO.12, and specifically comprises:

AATGCAAAAACCTTAGCAGAA(SEQ ID NO.12)

the nucleotide sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 3C1 is shown in SEQ ID NO.13, and specifically comprises:

CAACATCATTATGGTACTCCGTGGACG(SEQ ID NO.13)

the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 3C1 is shown in SEQ ID NO.14, and specifically comprises the following steps:

GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACATTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAACATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO.14)

the nucleotide sequence of the light chain of the monoclonal antibody 3C1 is shown as SEQ ID NO.15, and specifically comprises the following steps:

ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACATTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAACATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA(SEQ ID NO.15)

that is, the nucleotides of the light chain of monoclonal antibody 3C1 contained 381 bases.

The nucleotide sequence of the heavy chain variable region complementarity determining region 1(CDR1) of monoclonal antibody 3C1 is shown in SEQ ID NO.16, and specifically comprises the following steps:

AACTTTAGTGTTCAC(SEQ ID NO.16)

the nucleotide sequence of the heavy chain variable region complementarity determining region 2(CDR2) of monoclonal antibody 3C1 is shown in SEQ ID NO.17, and specifically comprises the following steps:

GTGATGTGGAGTGGTGGAAGCACAGACTATAATGCTGCTTTCATATCC(SEQ ID NO.17)

the nucleotide sequence of the heavy chain variable region complementarity determining region 3(CDR2) of monoclonal antibody 3C1 is shown in SEQ ID NO.18, and specifically comprises the following steps:

AGCGGTCCTTATTACTATTCTCTGGACTAC(SEQ ID NO.18)

the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3C1 is shown in SEQ ID NO.19, and specifically comprises the following steps:

CAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTTTAGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATGTGGAGTGGTGGAAGCACAGACTATAATGCTGCTTTCATATCCAGACTGAGCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACGGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGAAGCGGTCCTTATTACTATTCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO.19)

the nucleotide sequence of the heavy chain of the monoclonal antibody 3C1 is shown as SEQ ID NO.20, and specifically comprises the following steps:

ATGGCTGTCCTGGTGCTGCTCTTCTGCCTGGTGACATTCCCAAGCTGTGTCCTATCCCAGGTGCAGCTGAAGCAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAACTTTAGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATGTGGAGTGGTGGAAGCACAGACTATAATGCTGCTTTCATATCCAGACTGAGCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTTAAAATGAACGGTCTGCAAGCTGATGACACAGCCATATACTACTGTGCCAGAAGCGGTCCTTATTACTATTCTCTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO. 20). that is, the nucleotide of the heavy chain of monoclonal antibody 3C1 contains 411 bases.

2. Determination of ascites titer of monoclonal antibody

Designing a primer according to a BoTNF-alpha cDNA sequence (XM _005223596.4) in GenBank, and expressing a primer sequence P3: 5' -TTCTGTTCCAGGGGCCCCTG of a recombinant vector pGEX-6P-1-BoTNF-alphaGGATCCCTCAGGTCCTCTTCTCAAGCCTCAA-3 '(the underlined part is the enzyme cutting site of BamH I), P4: 5' -AGTCAGTCACGATGCGGCGCTCGAGTCACAGGGCGATGATCCCAAAGTAG (underlined is the Xho I cleavage site).

And carrying out double enzyme digestion on the pGEX-6P-1 vector, and recovering the purified vector. And uniformly mixing the purified vector and the BoTNF-alpha target gene according to the molar mass ratio of 1:3, adding one-step method ligase to act for 30min at 37 ℃, connecting the target gene and the vector, and thermally transferring the target gene and the vector into BL 21. The recombinant strain was named BL21(pGEX-6 p-1-BoTNF-. alpha.). Then recombinant bacteria BL21(pGEX-6 p-1-BoTNF-alpha) are induced, expressed and purified to obtain rGST-BoTNF-alpha purified protein.

rGST-BoTNF-. alpha.was diluted to 0.5. mu.g/mL using carbonate buffer, 100. mu.L per well was coated on ELISA plates, overnight at 4 ℃; PBST is washed for 3 times, 200 mu L of blocking solution is added into each hole, and incubation is carried out for 2h at 37 ℃; the monoclonal antibody ascites (the sample prepared in the first part of example 2) was diluted 100. mu.L per well in multiples, SP2/0 ascites was diluted in the same fold (the preparation method of SP2/0 ascites was referred to example 2 except that hybridoma cell 3C1 was replaced with SP2/0 cell) as a negative control, and incubated at 37 ℃ for 2 h; washing with PBST for 3 times, adding goat anti-mouse IgG enzyme-labeled secondary antibody with working concentration, incubating at 37 deg.C for 1.5h, wherein each well is 100 μ L; after PBST washing, TMB color development, determination of OD by enzyme-linked detector₄₅₀The value of (3) is determined by taking the P/N more than or equal to 2.1 as a determination standard.

The result shows that the titer of the monoclonal antibody mAb3C1 is 1: 13107200. The monoclonal antibody mAb3C1 has high potency, indicating that the use of the monoclonal antibody for preparing diagnostic reagents can produce high sensitivity.

3. Identification of monoclonal antibody specificity

The specificity of the monoclonal antibody is identified by adopting an indirect ELISA method. 0.5. mu.g/mL of commercial rBoTNF-. alpha. (R.USA)&D, 2279-BT-025), rBoIL-2(Kingfisher-Biotech, rp0026B-005), rBoIFN-. gamma.&Company D, 2300-BG-02), rPoTNF-alpha (R.USA)&Company D, 690-PT-025), rMoTNF-. alpha.R&D, 485-MI-100), ShTNF- α (Abcam, UK, ab184579) and rhuTNF- α (PeproTech, 300-01A-50) proteins were coated overnight; the next day, PBST was washed 3 times for 5min each time, the liquid was patted dry as much as possible, and 2% bovine serum albumin BSA in phosphate buffered saline PBS was blocked for 2 h; PBST is washed for 3 times, each time is 5min, the liquid is dried as much as possible, culture supernatant of hybridoma cell 3C1 is added, 100 mu L/hole is formed, and incubation is carried out for 2h at 37 ℃; PBST is washed for 6 times, each time is 5min, the washing liquid is dried as much as possible, HRP-goat anti-mouse IgG diluted to the working concentration by PBS of 1% BSA is added, 100 mu L/hole is formed, and incubation is carried out for 1h at 37 ℃; PBST is washed for 6 times, each time is 5min, the washing solution is dried as much as possible, TMB color development solution is added, 100 mu L/hole is formed, and incubation is carried out for 5min in a dark place at 37 ℃; 2mol/L H was added₂SO₄The reaction was stopped, 50. mu.L/well and OD was determined₄₅₀The value is obtained.

In indirect ELISA test, monoclonal antibody MAb3C1 only reacts with commercial rBoTNF-alpha and ShTNF-alpha, but not with other prokaryotic expression recombinant cytokines, and the specific results are shown in Table 1, which shows that the monoclonal antibody secreted by the hybridoma cell strain has cross-reaction of cattle and sheep, and the anti-TNF-alpha monoclonal antibody has good reactivity, specificity and affinity with rBoTNF-alpha and ShTNF-alpha proteins.

TABLE 1 identification of monoclonal antibody specificity

Example 4: assembly of Western-Blot kit

The Western-blot kit comprises the following assembly steps:

1. preparing a horse radish peroxidase-labeled anti-TNF-alpha monoclonal antibody (named HRP-3C1) with bovine and ovine cross reaction:

weighing 2mg of horseradish peroxidase (HRP) in a 5mL centrifuge tube, dissolving with 0.5mL deionized water, and storing the solution in dark place immediately after the solution is reddish brown; adding 0.5mL of freshly prepared 0.06mol/L sodium periodate, changing the solution into cyan at the moment, and keeping away from light at 4 ℃ for 30 min; adding 160mmol/L ethylene glycol 0.5mL, and reacting at room temperature for 30 min; adding purified monoclonal antibody MAb3C 12 mg, mixing, placing the mixture into dialysis bag, dialyzing in 2L 0.05mol/L carbonate buffer solution, and standing at 4 deg.C overnight; transferring the dialysate into a centrifuge tube, adding 0.2mL of freshly prepared 5mg/mL sodium borohydride, uniformly mixing, and acting at 4 ℃ for 2 h; adding equal volume of saturated ammonium sulfate, acting at 4 deg.C for 30min, centrifuging at 4000rpm/min for 20min, discarding supernatant, dissolving precipitate with PBS, dialyzing at 4 deg.C overnight, adding equal volume of glycerol, adding 0.1% ProClin300, filtering with 0.45 μm filter membrane, packaging at a volume of 110 μ L/piece, and storing at 2-8 deg.C.

2. The kit is prepared by packaging and assembling horseradish peroxidase-labeled anti-TNF-alpha monoclonal antibody MAb3C 1(HRP-3C1) with bovine and ovine cross reaction, PMA and ionomycin stimulant.

Furthermore, the kit is sequentially assembled with: one or more of sealing liquid (TBST containing 5% skimmed milk powder), washing liquid (TBST) and substrate liquid (DAB developing liquid).

Example 5: Western-Blot detection of recombinant bovine and ovine TNF-alpha protein

1. Expression and purification of ShTNF-alpha protein:

designing a primer according to the ShTNF-alpha cDNA sequence (NM-001024860) in GenBank, and expressing the primer sequence P5: 5' -GCTGATATCGGATCC of the recombinant vector pET-30a (+) -ShTNF-alphaGAATTCGGGACACCAGGGGACCAG-3 '(the underlined part is the restriction site of EcoR I) P6: 5' -GTGGTGGTGGTGGTGCTCGAGTTTTTTTTTTTTTTTCTTTTCTCAGG-3' (underlined part is the Xhol I cleavage site). Primer sequence P7: 5' -CCCCTGGGATCCCCG of expression recombinant vector pGEX-6P-1-ShTNF-alphaGAATTCGGGACACCAGGGGACCAG-3 '(the underlined part is the restriction site of EcoR I), P8: 5' -GTCACGATGCGGCCGCTCGAGTTTTTTTTTTTTTTTCTTTTCTCAGG (underlined is the Xho I cleavage site).

Total bovine peripheral blood lymphocyte RNA was extracted and reverse transcribed according to the RNeasy plus Mini kit instructions. The ShTNF-alpha gene is amplified and the pET-30a (+) and pGEX-6p-1(+) vectors are subjected to double enzyme digestion. Mixing the recovered and purified vector and the target gene at a molar mass ratio of 1:3, adding one-step method ligase, reacting at 37 ℃ for 30min, connecting the target gene and the vector, and performing heat transfer to BL21(DE 3). The recombinant bacteria are respectively named as BL21(DE3) (pET-30a (+) -ShTNF-alpha) and BL21(pGEX-6 p-1-ShTNF-alpha). Then, recombinant bacteria BL21(DE3) (pET-30a (+) -ShTNF-alpha) and BL21(pGEX-6 p-1-ShTNF-alpha) are induced, expressed and purified to obtain purified rHis-ShTNF-alpha and rGST-ShTNF-alpha recombinant proteins.

2. SDS-PAGE electrophoresis

The recombinant proteins rHis-BoTNF-alpha, rGST-BoTNF-alpha, rHis-ShTNF-alpha and rGST-ShTNF-alpha are added to a protein loading buffer respectively, boiled at 100 ℃ for 10min and subjected to SDS-PAGE electrophoresis.

3. Transfer printing

After electrophoresis is finished, the gel is placed in a transfer Buffer to be soaked for 5min, meanwhile, the NC membrane and the filter paper are soaked in the same way, 2 pieces of filter paper-NC membrane-gel-2 pieces of filter paper are laid in the sequence from top to bottom, then the gel is transferred to a semi-dry transfer printing instrument, and air bubbles are removed. Proteins were transferred to NC membranes by a pyxis (tm) Gel Processor rapid transfer printer.

4. Detection of

Sealing with PBS containing 5% skimmed milk, shaking at room temperature and sealing overnight;

washing the membrane by PBST for 4 times, and drying the residual washing liquid on the membrane as much as possible after 10min each time;

③ adding HRP-3C1 (prepared in example 4) diluted with PBS1: 1000, and shaking at room temperature for 2 h;

cleaning the membrane by PBST for 4 times, and drying the residual cleaning solution on the membrane as much as possible for 10min each time; and after color development, photographing a reaction result by a scanner.

The results are shown in FIG. 1, A shows a Western-Blot result chart of mAb3C1 reacting with rHis-BoTNF-alpha and rGST-BoTNF-alpha, and B shows a Western-Blot result chart of mAb3C1 reacting with rHis-ShTNF-alpha and rGST-ShTNF-alpha. The result shows that the Western-Blot method can effectively detect the BoTNF-alpha and ShTNF-alpha proteins expressed by an escherichia coli expression system and has better sensitivity.

Example 6: Western-Blot detection of bovine and sheep peripheral blood mononuclear cell samples

1. Preparation of peripheral blood mononuclear cells of cattle and sheep

Adding 5mL of cattle and sheep blood to be detected into a heparin sodium-containing blood collection tube aseptically, and inverting and uniformly mixing the collected blood to obtain anticoagulation blood;

secondly, diluting the anticoagulated blood and the sterilized PBS by 1:1, slowly adding the diluted cattle and sheep blood into a sterile centrifuge tube containing lymphocyte separation fluid according to the proportion of 1:1 to form an obvious interface, and centrifuging for 20-30min at room temperature of 2000 rmp;

thirdly, the peripheral blood mononuclear cells exist in the cloud-mist layer, the peripheral blood mononuclear cell layer is absorbed into a clean centrifugal tube by a sterilizing dropper, sterilized PBS is added, the cells are evenly mixed, then the cells are centrifuged at 2000rmp at 4 ℃ for 10min, and the centrifugation is repeated twice to obtain precipitated cells;

fourthly, removing the supernatant culture solution, adding complete 1640 culture medium to resuspend the precipitated cells, taking 10 mu L of cell suspension, adding 10 mu L of phenol blue to mix evenly, adding the cell suspension into a blood counting plate, counting under a microscope, and diluting the cell suspension to 1 × 10 by using the complete 1640 culture medium⁷Individual cells/mL.

2. Cell incubation

The following reagents were added to 24-well cell culture plates: adding 500 μ L diluted cell suspension into each well, adding PMA with concentration of 500ng/mL and ionomycin with final concentration of 1 μ g/mL for stimulation, standing at 37 deg.C and 5% CO₂Culturing in an incubator for 24-48 hours, and collecting cells as a detection sample;

3. SDS-PAGE electrophoresis

Adding the cell lysate into protein loading buffer, boiling at 100 deg.C for 10min, and performing SDS-PAGE electrophoresis.

4. Transfer printing

5. Detection of

The result shows that the Western-Blot detection kit can effectively detect the natural TNF-alpha protein of the cattle and sheep secreted by the peripheral blood mononuclear cells of the cattle and sheep.

Example 7: assembly of direct immunofluorescence (DFA) detection kit

The assembly procedure for the DFA kit was as follows:

1. preparing fluorescein isothiocyanate labeled anti-TNF-alpha monoclonal antibody (named FITC-3C1) with cattle and sheep cross reaction:

the purified MAb3C1 monoclonal antibody was labeled using a standard fluorescein isothiocyanate labeling protocol. Dialyzing the crosslinking reaction liquid for three times at 4 ℃ until the pH is 9.0 by using monoclonal antibody MAb3C1 (the concentration is more than or equal to 1mg/ml) to be crosslinked; dissolving freshly prepared FITC (concentration of 1mg/mL) in DMSO; according to P: f (protein: FITC) ═ 1 mg: slowly adding FITC into the antibody solution at a ratio of 150 μ g, gently shaking while adding to uniformly mix the FITC and the antibody, and reacting for 8h at 4 ℃ in a dark place; adding 5mol/L NH₄Cl to the final concentration of 50mmol/L, and terminating the reaction at 4 ℃ for 2 h; dialyzing the cross-linked substance in PBS for more than four times until the dialyzate is clear; identifying the concentration of the protein of the cross-linked substance and the F/P ratio; FITC-crosslinked protein should be placed in phosphate buffer at pH7.4, and 0.1% NaN added₃1% BSA, stored at 4 ℃ in the dark.

2. An anti-TNF-alpha monoclonal antibody (FITC-3C1) with bovine and ovine cross reaction marked by fluorescein isothiocyanate, a stimulant PMA and ionomycin are respectively packaged and assembled into a kit.

Further, the kit is assembled with: one or more of a fixing agent (paraformaldehyde) and a washing solution.

Example 8: direct immunofluorescence (DFA) detection of TNF-alpha of cattle and sheep

1. Construction of recombinant plasmids pCMV-Myc-BoTNF-alpha and pCMVP-Myc-ShTNF-alpha

Carrying out double enzyme digestion on the pCMV-Myc vector, recovering a purified vector, uniformly mixing the vector and target genes (BoTNF-alpha and ShTNF-alpha) according to the molar mass ratio of 1:3, adding one-step method ligase, acting for 30min at 37 ℃, and connecting the target genes and the vector. The recombinant plasmids are named as pCMV-Myc-BoTNF-alpha and pCMVP-Myc-ShTNF-alpha respectively.

2. Transfection

24h before transfection, Hela cells are digested and inoculated in a 24-well plate at 2X 10⁵Individual cells/well;

② add

1.5 μ L into 1.5mL finger tubes containing Opti-MEM to a total volume of 50 μ L;

thirdly, 1 mu L of each recombinant plasmid pCMV-Myc-BoTNF-alpha, pCMVP-Myc-ShTNF-alpha and empty vector plasmid pCMV-Myc and P3000 are respectively added^TM2 μ L to 1.5mL finger tubes containing Opti-MEM to a total volume of 50 μ L;

fourthly, respectively adding

50 mu L of Opti-MEM, standing for 5min, adding into the cell plate, and placing in an incubator for continuous culture.

3. Detection of eukaryotic expression bovine and ovine TNF-alpha protein

Removing culture supernatants of Hela (pCMV-Myc-BoTNF-alpha), Hela (pCMV-Myc-ShTNF-alpha) and Hela (pCMV-Myc) cells by suction, washing twice by using 500 mu L PBS, adding 500 mu L of ice methanol precooled at the temperature of 20 ℃ below zero, and fixing for 10min at the temperature of 20 ℃ below zero;

② washing with 500 μ L PBS for three times, adding MAb FITC-3C1 (prepared in example 7) diluted at a ratio of 1:1000, and incubating for 2h in a water bath at 37 ℃;

③ washing with 500. mu.L PBS 3 times, and observing with a fluorescence inverted microscope.

The results show that the direct immunofluorescence (DFA) method can effectively detect the TNF-alpha protein of cattle and sheep in a eukaryotic expression system.

Example 9: direct immunofluorescence (DFA) detection of bovine and ovine peripheral blood mononuclear cell samples

1. Preparation of peripheral blood mononuclear cells of cattle and sheep

secondly, diluting the anticoagulated blood and the sterilized PBS1:1, slowly adding the diluted cattle and sheep blood into a sterile centrifuge tube containing lymphocyte separation fluid according to the proportion of 1:1 to form an obvious interface, and centrifuging for 20-30min at room temperature of 2000 rmp;

2. Cell incubation

The following reagents were added to 24-well cell culture plates: mu.L of culture medium was applied to each control well, and 500. mu.L of culture medium containing 500ng/mL PMA and 1. mu.g/mL ionomycin was applied to each positive well. Add 500. mu.L of diluted cell suspension to each well and incubate at 37 ℃ with 5% CO₂Culturing in an incubator for 24-48 hours, and collecting cells;

3. immunofluorescence detection of natural cattle and sheep TNF-alpha

Firstly, sucking out the culture supernatant of cells, washing twice by using 500 mu L PBS, adding 500 mu L of ice methanol precooled at the temperature of 20 ℃ below zero, and fixing for 10min at the temperature of 20 ℃ below zero;

② washing with 500 μ L PBS for three times, adding FITC-3C1 (prepared in example 7) diluted by 1:1000, and incubating for 2h in a water bath at 37 ℃;

The results are shown in FIG. 2, wherein the positive sample well has a distinct green fluorescence, while the control sample well has no green fluorescence, when testing bovine peripheral blood mononuclear cell samples, and the specific results are shown in FIG. 2(A, B). When the sheep peripheral blood mononuclear cell sample is detected, the positive sample hole has obvious green fluorescence, and the control sample hole has no green fluorescence, as shown in figure 2(C, D), which shows that the kit can effectively detect the cells of TNF-alpha in peripheral blood of cattle and sheep stimulated by PMA combined with ionomycin.

Example 10: assembly of TNF-alpha FCM detection kit for cattle and sheep

The FCM detection kit is assembled by the following steps:

1. preparation of fluorescein isothiocyanate-labeled anti-TNF-. alpha.monoclonal with bovine and ovine cross-reactivity (named FITC-3C 1):

the purified monoclonal antibody mAb3C1 was labeled using standard fluorescein isothiocyanate labeling. To-be-crosslinked monoclonal antibody mAb3C1 (concentration)>1mg/mL) was dialyzed against the crosslinking reaction solution three times at 4 ℃ until the pH was 9.0; freshly prepared FITC (at a concentration of 1mg/mL) was dissolved in DMSO; as per P: F (protein: FITC) ═ 1 mg: slowly adding FITC into the antibody solution at a ratio of 150 μ g, gently shaking while adding to uniformly mix the FITC and the antibody, and reacting for 8 hours at 4 ℃ in a dark place; adding 5mol/L NH₄Cl to the final concentration of 50mmol/L, and terminating the reaction at 4 ℃ for 2 h; dialyzing the cross-linked substance in PBS for more than four times until the dialyzate is clear; identifying the concentration of the protein of the cross-linked substance and the F/P ratio; FITC-crosslinked protein should be placed in phosphate buffer pH7.4, and 0.1% NaN added₃1% BSA, stored at 4 ℃ in the dark.

2. The anti-TNF-alpha monoclonal antibody with the cross reaction of cattle and sheep marked by fluorescein isothiocyanate and the PMA are combined with the ionomycin stimulant and are respectively packaged to assemble the kit.

Furthermore, the kit is sequentially assembled with: blocking agent (Brefeldin A Solution), fixative (paraformaldehyde), membrane disruption agent (Intracellular stabilization hybridization Wash Buffer), and washing Solution (1% BSA in PBS).

Example 11: FCM detection of bovine and sheep peripheral blood mononuclear cell samples

1. Preparation of peripheral blood mononuclear cells of cattle and sheep

Adding 5mL of peripheral blood of a cow or a sheep to be detected into a blood collection tube containing heparin sodium in an aseptic manner, and inverting and uniformly mixing the collected blood to obtain anticoagulation blood;

2. Cell incubation

Adding the following reagents into a 24-well cell culture plate: mu.L of culture medium was applied to each control well, and 500. mu.L of culture medium containing 500ng/mL PMA and 1. mu.g/mL ionomycin was applied to each positive well. Add 500. mu.l of cell suspension per well at 37 ℃ in 5% CO₂Culturing in an incubator for 4-6 hours;

② adding cell factor secretion blocking agent BFA, placing at 37 deg.C and 5% CO₂The incubator was incubated for 16 hours.

3. And (3) detecting cytokines:

collecting cultured cells the next day, washing the cells with PBS containing 1% BSA, centrifuging at 4 ℃ and 2000rpm for 10min, and discarding the supernatant;

② fixing agent, standing at room temperature for 15 min; cells were washed with PBS containing 1% BSA; centrifuging at 4 deg.C and 2000rpm for 10min, discarding supernatant, and repeating twice;

③ diluting FITC-labeled anti-TNF-alpha monoclonal antibody FITC-3C1 (prepared in example 10) with a film-breaking agent and having cross reaction with cattle and sheep to 0.5 mu g/mL, standing at room temperature for 15min, and washing cells with PBS containing 1% BSA; centrifuging at 4 deg.C and 2000rpm for 10min, discarding supernatant, and repeating twice;

and fourthly, resuspending the cells by using 200 mu L of PBS, and detecting the proportion of the cells secreting the TNF-alpha of the cattle and the sheep by FACS.

The results are shown in FIG. 3, and the results show that when bovine and sheep peripheral blood mononuclear cell samples are detected, the proportion (18.4%) of secreted bovine TNF-alpha cells in the positive sample well B is higher than that in the control sample well (0.051%), and the proportion (13.4%) of secreted sheep TNF-alpha cells in the positive sample well D is higher than that in the control sample well (0.027%), which indicates that the kit can effectively detect PMA combined with ionomycin to stimulate the cells secreted bovine and sheep TNF-alpha in the peripheral blood of the bovine and sheep.

Example 12: assembly of FCM detection kit for bovine (ovine) tuberculosis

Preparation of fluorescein isothiocyanate-labeled anti-TNF-. alpha.monoclonal with bovine and ovine cross-reactivity (named FITC-3C 1):

The staining plate, the fluorescein isothiocyanate labeled anti-TNF-alpha monoclonal 3C1 with bovine and ovine cross reaction, the fusion protein of specific stimulators CFP-10 and ESAT-6, the negative control (complete 1640 culture medium) and the positive control (PMA and ionomycin) are respectively packaged and assembled into a kit.

Further, the kit is assembled with: fixative (paraformaldehyde), disrupting agent (Intracellular stabilization filtration Wash Buffer), detergent (1% BSA in PBS), cell culture fluid (complete 1640), Staining plate, flow tube.

Example 13: bovine (sheep) tuberculosis FCM detection kit for detecting bovine and sheep peripheral blood mononuclear cell samples

1. Preparing peripheral blood mononuclear cells of cattle and sheep

Adding 5mL of peripheral blood of each of tuberculosis positive cattle, tuberculosis negative cattle, tuberculosis positive sheep and tuberculosis negative sheep into a blood collection tube containing heparin sodium under aseptic condition, and reversing and uniformly mixing the collected blood to obtain anticoagulation blood;

secondly, diluting the anticoagulated blood and the sterilized PBS by 1:1, slowly adding the diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation fluid according to the ratio of 1:1 to form an obvious interface, and centrifuging for 20-30min at room temperature of 2000 rmp;

2. Cell incubation

Adding the following reagents into a 24-well cell culture plate: mu.L of culture broth to each control well, 500. mu.L of culture broth containing 10. mu.g/mL of CFP-10 and ESAT-6 fusion protein to each positive well. Culturing at 37 deg.C in 5% CO2 incubator for 4-6 hr;

② adding cell factor secretion blocking agent BFA, placing them into 37 deg.C culture box and 5% CO2 culture box and culturing for 16 hr.

3. And (3) detecting cytokines:

③ diluting an FITC-labeled anti-TNF-alpha monoclonal antibody MAb FITC-3C1 (prepared in example 12) with a film-breaking agent and having a bovine and ovine cross reaction to 0.5 mu g/mL, standing at room temperature for 15min, and washing cells with PBS containing 1% BSA; centrifuging at 4 deg.C and 2000rpm for 10min, discarding supernatant, and repeating twice;

As shown in FIG. 4, it can be seen from FIG. 4 that the ratio of TNF-alpha cells secreted from bovine tuberculosis-positive cattle after being stimulated by CFP-10 and ESAT-6 fusion protein is higher than that of TNF-alpha cells secreted from bovine tuberculosis-negative cattle when detecting bovine peripheral blood mononuclear cell samples. After being stimulated by the CFP-10 and ESAT-6 fusion protein, the TNF-alpha cell ratio of the goat secreted by the tuberculosis-positive sheep is higher than that of the goat secreted by the tuberculosis-negative sheep. The result shows that the kit can effectively distinguish the cattle with positive negative tuberculosis and the sheep with positive negative tuberculosis, and has better sensitivity.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

And (3) classification and naming: hybridoma cell line 3C1

The preservation unit is called and abbreviated as: china Center for Type Culture Collection (CCTCC)

Address: wuhan university of Wuhan, China

The preservation date is as follows: 6 month and 17 days 2019

The preservation number is: CCTCC NO: c2019129

Sequence listing

<110> Yangzhou university

<120> preparation and application of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

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Arg Ala Ser Glu Asn Ile Tyr Ile Tyr Leu Ala

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<213> Artificial Sequence (Artificial Sequence)

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Asn Ala Lys Thr Leu Ala Glu

1 5

<210> 3

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gln His His Tyr Gly Thr Pro Trp Thr

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<213> Artificial Sequence (Artificial Sequence)

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Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

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Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ile Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val

35 40 45

Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Phe Ser Leu Asn Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 5

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ser Val Pro Thr Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr

1 5 10 15

Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser

20 25 30

Ala Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn

35 40 45

Ile Tyr Ile Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro

50 55 60

Gln Leu Leu Val Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Phe Ser Leu Asn Ile Asn

85 90 95

Ser Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr

100 105 110

Gly Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 6

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asn Phe Ser Val His

1 5

<210> 7

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile Ser

1 5 10 15

<210> 8

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Ser Gly Pro Tyr Tyr Tyr Ser Leu Asp Tyr

1 5 10

<210> 9

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Phe

20 25 30

Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile

50 55 60

Ser Arg Leu Ser Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Asn Gly Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ser Gly Pro Tyr Tyr Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 10

<211> 137

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Ala Val Leu Val Leu Leu Phe Cys Leu Val Thr Phe Pro Ser Cys

1 5 10 15

Val Leu Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln

20 25 30

Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu

35 40 45

Thr Asn Phe Ser Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

50 55 60

Glu Trp Leu Gly Val Met Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala

65 70 75 80

Ala Phe Ile Ser Arg Leu Ser Ile Ile Lys Asp Asn Ser Lys Ser Gln

85 90 95

Val Phe Phe Lys Met Asn Gly Leu Gln Ala Asp Asp Thr Ala Ile Tyr

100 105 110

Tyr Cys Ala Arg Ser Gly Pro Tyr Tyr Tyr Ser Leu Asp Tyr Trp Gly

115 120 125

Gln Gly Thr Ser Val Thr Val Ser Ser

130 135

<210> 11

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgagcaagtg agaatattta catttattta gca 33

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aatgcaaaaa ccttagcaga a 21

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

caacatcatt atggtactcc gtggacg 27

<210> 14

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60

atcacatgtc gagcaagtga gaatatttac atttatttag catggtatca gcagaaacag 120

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180

aggttcagtg gcagtggatc aggcacacag ttttctctga acatcaacag cctgcagcct 240

gaagattttg ggagttatta ctgtcaacat cattatggta ctccgtggac gttcggtgga 300

ggcaccaagc tggaaatcaa a 321

<210> 15

<211> 381

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgagtgtgc ccactcaggt cctggggttg ctgctgctgt ggcttacagg tgccagatgt 60

gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 120

atcacatgtc gagcaagtga gaatatttac atttatttag catggtatca gcagaaacag 180

ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 240

aggttcagtg gcagtggatc aggcacacag ttttctctga acatcaacag cctgcagcct 300

gaagattttg ggagttatta ctgtcaacat cattatggta ctccgtggac gttcggtgga 360

ggcaccaagc tggaaatcaa a 381

<210> 16

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

aactttagtg ttcac 15

<210> 17

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gtgatgtgga gtggtggaag cacagactat aatgctgctt tcatatcc 48

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

agcggtcctt attactattc tctggactac 30

<210> 19

<211> 354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

caggtgcagc tgaagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccatc 60

acctgcacag tctctggttt ctcattaact aactttagtg ttcactgggt tcgccagcct 120

ccaggaaagg gtctggagtg gctgggagtg atgtggagtg gtggaagcac agactataat 180

gctgctttca tatccagact gagcatcatc aaggacaact ccaagagcca agttttcttt 240

aaaatgaacg gtctgcaagc tgatgacaca gccatatact actgtgccag aagcggtcct 300

tattactatt ctctggacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354

<210> 20

<211> 411

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

atggctgtcc tggtgctgct cttctgcctg gtgacattcc caagctgtgt cctatcccag 60

gtgcagctga agcagtcagg acctggccta gtgcagccct cacagagcct gtccatcacc 120

tgcacagtct ctggtttctc attaactaac tttagtgttc actgggttcg ccagcctcca 180

ggaaagggtc tggagtggct gggagtgatg tggagtggtg gaagcacaga ctataatgct 240

gctttcatat ccagactgag catcatcaag gacaactcca agagccaagt tttctttaaa 300

atgaacggtc tgcaagctga tgacacagcc atatactact gtgccagaag cggtccttat 360

tactattctc tggactactg gggtcaagga acctcagtca ccgtctcctc a 411

Claims

1. A hybridoma cell strain with a preservation number of CCTCC NO: C2019129.

2. an anti-TNF-alpha antibody is prepared from a polypeptide with a preservation number of CCTCC NO: c2019129.

3. An anti-TNF-alpha antibody comprises a heavy chain variable region and a light chain variable region, wherein the CDR of the light chain variable region is CDR-L1 with an amino acid sequence shown in SEQ ID No.1, CDR-L2 with an amino acid sequence shown in SEQ ID No.2 and CDR-L3 with an amino acid sequence shown in SEQ ID No.3, and the CDR of the heavy chain variable region is CDR-H1 with an amino acid sequence shown in SEQ ID No.6, CDR-H2 with an amino acid sequence shown in SEQ ID No. 7and CDR-H3 with an amino acid sequence shown in SEQ ID No. 8.

4. The anti-TNF- α antibody of claim 3, wherein the amino acid sequence of the light chain variable region of the anti-TNF- α antibody comprises the amino acid sequence set forth in SEQ ID No. 4.

5. The anti-TNF- α antibody of claim 3, wherein the amino acid sequence of the light chain of the anti-TNF- α antibody comprises the amino acid sequence set forth in SEQ ID No. 5.

6. The anti-TNF- α antibody of claim 3, wherein the amino acid sequence of the heavy chain variable region of the anti-TNF- α antibody comprises the amino acid sequence set forth in SEQ ID No. 9.

7. The anti-TNF- α antibody of claim 3, wherein the amino acid sequence of the heavy chain of the anti-TNF- α antibody comprises the amino acid sequence set forth in SEQ ID No. 10.

8. An isolated polynucleotide encoding the anti-TNF- α antibody of any one of claims 3-7.

9. A construct comprising the isolated polynucleotide of claim 8.

10. An antibody expression system comprising the construct or genome of claim 9 integrated with an exogenous polynucleotide of claim 8.

11. A method of producing an anti-TNF- α antibody as claimed in any of claims 2 to 7, comprising the steps of: culturing the antibody expression system of claim 10 to express said antibody, and purifying and isolating said antibody; and/or the expression vector is expressed by a collection number of CCTCC NO: hybridoma production of C2019129.

12. Use of an anti-TNF- α antibody of any of claims 2 to 7 in the preparation of a TNF- α detection kit.

13. A test kit comprising an anti-TNF- α antibody of any of claims 2-7.