CN110760483A

CN110760483A - Preparation and application of anti-TNF- α monoclonal antibody with cattle and sheep cross reaction

Info

Publication number: CN110760483A
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: 2020-02-07
Anticipated expiration: 2039-11-08
Also published as: CN110760483B

Abstract

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

Description

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

Technical Field

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

Background

After the immune system of the organism is activated, a cell factor which can lead the tumor to generate hemorrhagic necrosis is generated, and is named as tumor necrosis factor α (TNF- α). TNF- α is a cell factor related to systemic inflammation and is mainly secreted by macrophages, and the cell factor has the main function of regulating immune cells, is used as an endogenous pyrogen and can cause fever, cause apoptosis, prevent tumorigenesis, virus replication and the like.

The immunological detection of TNF- α is an experimental method which is established on the basis of a specific anti-TNF- α monoclonal antibody (MAb) and is used for qualitative and quantitative analysis of TNF- α in a sample, and a plurality of detection methods can be established by applying different monoclonal antibody markers and detection technologies, such as an Enzyme-linked Immunosorbent Assay (ELISA) and a Flow Cytometry (FCM) analysis method and the like.

However, the anti-bovine and anti-ovine TNF- α antibodies prepared in the prior art are often applied to the detection method, and particularly have poor effects when being 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 application 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- α antibody, which is produced by a hybridoma cell strain or a passage cell strain thereof with the preservation number of CCTCC NO: C2019129.

In another aspect, the present invention provides an anti-TNF- α antibody, wherein the anti-TNF- α antibody comprises a heavy chain variable region and a light chain variable region, the CDR of the light chain variable region comprises CDR-L1 with the amino acid sequence as shown in SEQ ID No.1, CDR-L2 with the amino acid sequence as shown in SEQ ID No.2, and CDR-L3 with the amino acid sequence as shown in SEQ ID No.3, and the CDR of the heavy chain variable region comprises CDR-H1 with the amino acid sequence as shown in SEQ ID No.6, CDR-H2 with the amino acid sequence as shown in SEQ ID No.7, and CDR-H3 with the amino acid sequence as shown 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 comprises the amino acid sequence set forth in 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 comprises the amino acid sequence set forth in SEQ ID No. 10.

In another aspect, the present invention provides an isolated polynucleotide encoding the heavy chain variable region and/or the light chain variable region and/or the 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.

According to another aspect of the present invention, there is provided a method for producing the anti-TNF- α antibody, comprising the steps of culturing an expression system for the antibody to express the antibody, purifying and isolating the 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- α antibody in preparing a TNF- α detection kit.

In another aspect, the invention provides a test kit comprising the anti-TNF- α antibody.

Drawings

FIG. 1 is a graph showing the Western-Blot detection results of the proteins of cattle and sheep TNF- α, rHis-BoTNF- α and rGST-BoTNF- α, and B.rHis-ShTNF- α and rGST-ShTNF- α, in the Western-Blot method of example 5.

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- α antibody prepared by the hybridoma cell strain, wherein the anti-TNF- α antibody can be combined with natural bovine and ovine TNF- α 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 anti-TNF- α antibody provided by the invention can be secreted from the hybridoma cell strain provided by the first aspect of the invention or a subcultured cell strain thereof, is usually a monoclonal antibody, generally refers to a population of antibodies, wherein the antibodies included in the population are basically the same (except for a few naturally occurring mutations which may exist), the monoclonal antibody is generally directed to a specific determinant on an antigen, the anti-TNF- α antibody can be of murine origin, the anti-TNF- α antibody can be combined with natural bovine and ovine TNF- α with high affinity, and only reacts with rBoTNF- α and ShTNF- α (natural TNF- α of bovine and ovine) in the specificity identification of the antibody, but does not react with other expressed recombinant cytokines, and has the characteristics of high potency, high sensitivity and high specificity in the measurement.

In a third aspect, the invention provides an anti-TNF- α antibody, wherein the anti-TNF- α antibody comprises a heavy chain variable region and a light chain variable region, the CDR of the light chain variable region comprises 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 comprises 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.

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 comprise:

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 as 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 as 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 can 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 can 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 preparing the anti-TNF- α antibody of the second or third aspect of the present invention, which comprises culturing an expression system of the antibody of claim 11 under conditions suitable for expression of the antibody, thereby expressing the antibody, and purifying and isolating the antibody, the conditions suitable for expression of the antibody should be known to those skilled in the art, and those skilled in the art can select a suitable medium based on experience, culturing under conditions suitable for growth of the host cells, inducing the selected promoter by a suitable method (such as temperature shift or chemical induction) after the host cells have grown to a suitable cell density, and culturing the cells for a further period of time.

The anti-TNF- α antibody may also be produced from a hybridoma having a collection number of CCTCC NO: C2019129, which may be prepared by in vivo induced ascites method, suitable methods for providing monoclonal antibodies by in vivo induced ascites using hybridomas are known to those skilled in the art, and the mice may be inoculated with hybridoma cells intraperitoneally by experience, and ascites collected, if necessary, and recombinant proteins may be isolated and purified by various separation methods using their physical, chemical, and other properties, which are well known to those skilled in the art.

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

The eighth aspect of the present invention provides a detection kit, which comprises the anti-TNF- α antibody provided in the second aspect or the third aspect, and particularly can be in a diagnostically effective dose, wherein the effective dose generally refers to an amount capable of providing a diagnostic benefit, so the diagnostic kit can generally diagnose the TNF- α antigen as a biomarker for a target TNF- α antigen, the diagnostic kit can further comprise a marker of an anti-TNF- α antibody, wherein the marker of the anti-TNF- α antibody can be generally used for marking the anti-TNF- α antibody, and the types of selectable markers include but are not limited to one or a combination of a fluorescent marker, a radioactive marker, an enzyme marker, a chemiluminescent marker and the like.

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

In another embodiment of the present invention, the kit can be a direct immunofluorescence (DFA) assay kit, which is used for a method for detecting TNF- α protein in cattle and sheep using Hela cells as an expression host, and can also be used for analyzing a sample of peripheral blood mononuclear cells in cattle and sheep.

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 bovine and ovine TNF- α, thereby performing research on immune status evaluation and disease diagnosis of an organism.

According to a ninth aspect of the invention, there is provided a method of detecting, by the anti-TNF- α antibody of the second or third aspect of the invention or the detection kit of the eighth aspect of the invention, bovine TNF- α and/or ovine TNF- α in a sample, wherein the detection may be for non-diagnostic therapeutic purposes, and the non-diagnostic purpose detection includes detection of recombinantly expressed bovine or ovine TNF- α, detection of ex vivo tissue, and epitope identification studies.

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- α and sheep TNF- α. the Western-Blot detection kit established based on the monoclonal antibody provided by the invention can effectively detect bovine and sheep TNF- α proteins expressed by an escherichia coli expression system, and can also detect natural bovine and sheep TNF- α proteins secreted by bovine and sheep Peripheral Blood Mononuclear Cells (PBMC). The direct immunofluorescence (DFA) detection kit established based on the monoclonal antibody provided by the invention can effectively detect bovine and sheep peripheral blood mononuclear cell samples and bovine and sheep TNF- α secreted by eukaryotic plasmid expression, and the FCM detection kit of bovine and sheep TNF- α established based on the monoclonal antibody provided by the invention can detect bovine and sheep peripheral blood mononuclear cells secreting high level and sheep TNF- α when detecting bovine and sheep peripheral blood mononuclear cell samples, thus the bovine and sheep peripheral blood mononuclear cells secreting high level can be conveniently detected, and the related operation kit provided by the invention can be widely used for researching non-immune diagnosis of bovine and sheep diseases and non- α.

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 Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, 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- α recombinant protein

Designing a primer according to a BoTNF- α cDNA sequence (XM _005223596.4) in GenBank, and expressing a primer sequence P1: 5' -ACAAGGCCATGGCTGATATC of a recombinant vector pET-30a (+) -BoTNF- αGGATCCCTCAGGTCCTCTTCTCAA-3 '(the underlined part is the BamHI cleavage site) P2: 5' -TGGTGCTCGAGTGCGGCCGCAAGCTTTCACAGGGCGATGATCCCAAAGTA-3' (underlined is the Hind III site).

Extracting total RNA of bovine peripheral blood lymphocytes according to the specification of an RNeasy plus Mini kit, carrying out reverse transcription, carrying out double enzyme digestion on a pET-30a (+) vector, uniformly mixing the recovered and purified vector and a target gene according to a molar mass ratio of 1:3, adding one-step method ligase to act for 30min at 37 ℃, connecting the target gene and the vector, carrying out heat transfer on the target gene to BL21(DE3), and carrying out induced expression and purification on recombinant bacteria BL21(DE3) (pET-30a (+) -BoTNF- α) after the recombinant bacteria BL21(DE3) (pET-30a (+) -BoTNF- α) are named, so as to obtain the rHis-BoTNF- α purified protein.

2. Animal immunization

The specific immunization program comprises the following steps of immunizing a BALB/c mouse for the first time, injecting 100 mu g of rHis-BoTNF- α purified protein fully emulsified by Freund's complete adjuvant into the abdomen at multiple points, injecting 100 mu g of purified protein fully emulsified by Freund's incomplete adjuvant into the abdomen at multiple points after 2 weeks for secondary immunization, injecting 100 mu g of purified protein without adjuvant into the abdomen after 2 weeks for third immunization, collecting blood after 7 days to measure the titer of serum antibody, and selecting a mouse with higher titer 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 was added(TMB) color development, determination of OD by enzyme-linked assay₄₅₀The 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: adding the hybridoma 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- α monoclonal antibody with bovine and ovine Cross-reactivity

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 rabbit anti-sheep enzyme labeled secondary antibody 50 μ L/well diluted at 1:5000, washing for 3 times at 37 deg.C for 15 min; 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) that is, the heavy chain of monoclonal antibody 3C1 contains 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 primers according to BoTNF- α cDNA sequence (XM _005223596.4) in GenBank, and expressing primer sequence P3: 5' -TTCTGTTCCAGGGGCCCCTG of recombinant vector pGEX-6P-1-BoTNF- αGGATCCCTCAGGTCCTCTTCTCAAGCCTCAA-3 '(the underlined part is the enzyme cutting site of BamH I), P4: 5' -AGTCAGTCACGATGCGGCGCTCGAGTCACAGGGCGATGATCCCAAAGTAG (underlined is the Xho I cleavage site).

Uniformly mixing the purified vector and BoTNF- α target gene in a molar mass ratio of 1:3, adding one-step method ligase to act at 37 ℃ for 30min, connecting the target gene with the vector, carrying out heat transfer to BL21, naming the recombinant bacterium as BL21(pGEX-6P-1-BoTNF- α), and then carrying out induced expression and purification on the recombinant bacterium BL21(pGEX-6P-1-BoTNF- α) to obtain rGST-BoTNF- α purified protein.

rGST-BoTNF- α 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

Specificity of the monoclonal antibody was identified by indirect ELISA 0.5. mu.g/mL of commercial rBoTNF- α (R. U.S.A.)&D, 2279-BT-025), rBoIL-2(Kingfisher-Biotech, rp0026B-005), rBoIFN-. gamma.&Company D, 2300-BG-02), rPoTNF- α (U.S. R)&Company D, 690-PT-025), rMoTNF- α (U.S. R)&485-MI-100, ShTNF- α (Abcam, UK, ab184579) and rhuTNF- α (PeproTech, 300-01A-50) protein coating overnight, the following day, PBST washing 3 times, 5min each, draining the liquid as much as possible, blocking the phosphate buffer PBS of 2% bovine serum albumin BSA for 2h, PBST washing 3 times, 5min each, draining the liquid as much as possible, adding the culture supernatant of hybridoma 3C1, 100 uL/well, incubating at 37 ℃ for 2h, PBST washing 6 times, 5min each, draining the washing liquid as much as possible, adding HRP-goat anti-mouse IgG diluted to working concentration with PBS of 1% BSA, incubating at 100 uL/well, incubating at 37 ℃ for 1h, PBST washing 6 times, 5min each, draining the washing liquid as much as possible, adding TMB color developing solution, 100 uL/well, 37 ℃ for 5min, adding 2mol/L H min at 37 ℃ and keeping the washing liquid as dark₂SO₄The 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- α and ShTNF- α, 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 good reactivity, specificity and affinity with anti-TNF- α monoclonal antibody of cross reaction of cattle and sheep and rBoTNF- α and ShTNF- α 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- α monoclonal antibody (named as 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 12mg, 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 horse radish peroxidase-labeled anti-TNF- α 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 assay of recombinant bovine and ovine TNF- α proteins

1. Expression and purification of ShTNF- α protein:

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

Extracting total RNA of bovine peripheral blood lymphocytes according to the specification of an RNeasy plus Mini kit, carrying out reverse transcription, carrying out amplification on ShTNF- α gene, carrying out double enzyme digestion on pET-30a (+) and pGEX-6p-1(+) vectors, uniformly mixing the recovered and purified vectors and a target gene according to the molar mass ratio of 1:3, adding one-step method ligase for 30min at 37 ℃, connecting the target gene with the vectors, carrying out heat transfer to BL21(DE3), respectively naming recombinant bacteria BL21(DE3) (pET-30a (+) -ShTNF- α) and BL21(pGEX-6p-1-ShTNF- α) as recombinant bacteria, then carrying out induction expression and purification on recombinant bacteria BL21(DE3) (pET-30a (+) -ShTNF- α) and BL21(pGEX-6p-1-ShTNF- α) to obtain purified rHis-ShTNF- α and rHis-ShTNF-GST- α recombinant proteins.

2. SDS-PAGE electrophoresis

Recombinant proteins rHis-BoTNF- α, rGST-BoTNF- α, rHis-ShTNF- α and rGST-ShTNF- α were added to a protein loading buffer, 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

① blocked with 5% skim milk in PBS and blocked overnight at room temperature with shaking;

② washing the membrane with PBST for 4 times (10 min each time), and spin-drying the residual washing liquid on the membrane;

③ HRP-3C1 (prepared in example 4) diluted with PBS1: 1000 was added and shaken at room temperature for 2 h;

④ washing the membrane with PBST for 4 times (10 min each time), drying the residual washing liquid on the membrane as much as possible, developing color, and taking photo by scanner.

The result is shown in figure 1, A shows a Western-Blot result graph of mAb3C1 reacting with rHis-BoTNF- α and rGST-BoTNF- α, B shows a Western-Blot result graph of mAb3C1 reacting with rHis-ShTNF- α and rGST-ShTNF- α, and the result shows that the Western-Blot method can effectively detect the BoTNF- α and ShTNF- α 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

① sterile respectively adding 5mL of cattle and sheep blood to be tested into heparin sodium-containing blood collection tube, and mixing to obtain anticoagulation blood;

② diluting anticoagulant and sterilized PBS at a ratio of 1:1, slowly adding diluted sanguis bovis Seu Bubali and sanguis Caprae Seu Ovis into sterile centrifuge tube containing lymphocyte separation solution at a ratio of 1:1 to form obvious interface, and centrifuging at 2000rmp at room temperature for 20-30 min;

③ it can be seen that the peripheral blood mononuclear cells exist in the cloud-like layer, sucking the peripheral blood mononuclear cell layer with a sterilizing dropper to a clean centrifuge tube, adding sterilized PBS, mixing the cells uniformly, centrifuging at 4 deg.C 2000rmp for 10min, repeating twice to obtain precipitated cells;

④ discarding the supernatant culture solution, adding complete 1640 culture medium to resuspend the precipitated cells, adding 10 μ L of cell suspension into 10 μ L of phenol blue, mixing, adding into a blood counting plate, counting under a microscope, and diluting the cell suspension to 1 × 10 with complete 1640 culture medium⁷Individual cells/mL.

2. Cell incubation

The following assay was added to 24-well cell culture platesPreparation: 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 natural bovine and sheep TNF- α proteins secreted by peripheral blood mononuclear cells of bovine 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- α 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; freshly prepared FITC (concentration 1mg/mL) was dissolved 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- α monoclonal antibody (FITC-3C1) marked by fluorescein isothiocyanate and having bovine and ovine cross reaction, 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) assay of bovine and ovine TNF- α

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

The pCMV-Myc vector is subjected to double enzyme digestion and recovered and purified vector, the vector and target genes (BoTNF- α and ShTNF- α) are uniformly mixed according to the molar mass ratio of 1:3, one-step method ligase is added to act for 30min at 37 ℃, the target genes and the vector are connected, and recombinant plasmids are respectively named as pCMV-Myc-BoTNF- α and pCMVP-Myc-ShTNF- α.

2. Transfection

① before transfection, Hela cells were digested and inoculated in 24-well plates at 2X 10⁵Individual cells/well;

② is added

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

③ adding 1 μ L each of recombinant plasmid pCMV-Myc-BoTNF- α, pCMVP-Myc-ShTNF- α and empty vector plasmid pCMV-Myc, and P3000 ^TM2 μ L to 1.5mL finger tubes containing Opti-MEM to a total volume of 50 μ L;

④ respectivelyAdding into

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- α protein

① removing culture supernatant of Hela (pCMV-Myc-BoTNF- α), Hela (pCMV-Myc-ShTNF- α) and Hela (pCMV-Myc) cells, washing with 500 μ L PBS twice, adding 500 μ L of ice methanol pre-cooled at-20 deg.C, and fixing at-20 deg.C for 10 min;

② washed three times with 500 μ L PBS, added with 1:1000 diluted MAb FITC-3C1 (prepared in example 7), incubated in a 37 deg.C water bath for 2 h;

③ were washed 3 times with 500. mu.L PBS and observed using a fluorescent inverted microscope.

The results show that the direct immunofluorescence (DFA) method can effectively detect the TNF- α 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

② diluting anticoagulant and sterilized PBS1:1, slowly adding diluted cattle and sheep blood into a sterile centrifuge tube containing lymphocyte separation liquid according to the ratio of 1:1 to form an obvious interface, and centrifuging at 2000rmp for 20-30min at room temperature;

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 cow and sheep TNF- α

① removing the cell culture supernatant, washing with 500 μ L PBS twice, adding 500 μ L ice methanol pre-cooled to-20 deg.C, and fixing at-20 deg.C for 10 min;

② washed three times with 500 μ L PBS, added FITC-3C1 (prepared in example 7) diluted at 1:1000, incubated in a water bath at 37 deg.C for 2 h;

The result is shown in figure 2, when the bovine peripheral blood mononuclear cell sample is detected, the positive sample hole has obvious green fluorescence, and the control sample hole has no green fluorescence, and the specific result is shown in figure 2(A, B). when the ovine 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), the kit can effectively detect the PMA combined ionomycin-stimulated TNF- α cells in the bovine and ovine peripheral blood.

Example 10 Assembly of TNF- α 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- α monoclonal antibody 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: FITC was slowly added to the antibody solution at a rate of 150. mu.g, and mixed with the antibody by gently shakingUniformly reacting for 8 hours at the temperature of 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- α monoclonal antibody with cross reaction of cattle and sheep marked by fluorescein isothiocyanate and PMA are combined with an ionomycin stimulant and are packaged and assembled into a kit respectively.

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

① aseptic 5mL of peripheral blood of cattle or sheep to be detected is added into a blood collection tube containing heparin sodium, and after blood is collected, the mixture is inverted and mixed evenly to obtain anticoagulation blood;

2. Cell incubation

① the following reagents were added to a 24-well cell culture plate 50mu.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 cytokine secretion blocking agent BFA, adding 5% CO at 37 deg.C₂The incubator was incubated for 16 hours.

3. And (3) detecting cytokines:

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

② adding fixative, standing at room temperature for 15min, washing cells with PBS containing 1% BSA, centrifuging at 4 deg.C and 2000rpm for 10min, removing supernatant, and repeating twice;

③ diluting FITC-labeled anti-TNF- α monoclonal antibody FITC-3C1 (prepared in example 10) with cross reaction of cattle and sheep to 0.5 μ g/mL by using a membrane breaking agent, standing at room temperature for 15min, washing cells by using PBS containing 1% BSA, centrifuging at 2000rpm at 4 ℃ for 10min, discarding the supernatant, and repeating twice;

④ cells were resuspended in 200. mu.L PBS and FACS was performed to determine the proportion of cells secreting bovine and ovine TNF- α.

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 of secreted bovine TNF- α cells (18.4%) in the positive sample well B is higher than that of secreted bovine TNF- α cells (0.051%) in the control sample well (0.051%), and the proportion of secreted sheep TNF- α cells (13.4%) in the positive sample well D is higher than that of secreted bovine and sheep TNF- α cells in peripheral blood of bovine and sheep are effectively detected by the kit.

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

Preparation of fluorescein isothiocyanate-labeled anti-TNF- α monoclonal antibody 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.

The kit is prepared by packaging and assembling a staining plate, a fluorescein isothiocyanate labeled anti-TNF- α monoclonal 3C1 with bovine and ovine cross reaction, a fusion protein of specific stimulators CFP-10 and ESAT-6, a negative control (complete 1640 culture medium) and a positive control (PMA and ionomycin) respectively.

Further, the kit is assembled with: fixative (paraformaldehyde), disrupting agent (intracellular stabilization hybridization 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

① sterile 5mL of peripheral blood of the tuberculosis positive cattle, the tuberculosis negative cattle, the tuberculosis positive sheep and the tuberculosis negative sheep are respectively added into a blood collection tube containing heparin sodium, and after blood is collected, the mixture is inverted and mixed evenly to obtain anticoagulation blood;

② diluting anticoagulant and sterilized PBS at a ratio of 1:1, slowly adding diluted bovine blood into a sterile centrifuge tube containing bovine lymphocyte separation liquid at a ratio of 1:1 to form an obvious interface, and centrifuging at 2000rmp for 20-30min at room temperature;

④ discarding the supernatant culture solution, adding into the complete 1640 culture medium to resuspend the precipitated cells, adding 10 μ L cell suspension10 μ L of phenol blue was mixed well and added to a hemocytometer, counted under a microscope, and the cell suspension was diluted to 1X 10 using complete 1640 medium⁷Individual cells/mL.

2. Cell incubation

① Add the following reagents to a 24 well cell culture plate, 500. mu.L of culture to each control well, 500. mu.L of culture containing 10. mu.g/mL of the CFP-10 and ESAT-6 fusion proteins to each positive well, incubate at 37 ℃ for 4-6 hours in a 5% CO2 incubator;

② adding cytokine secretion blocking agent BFA, and culturing in 5% CO2 incubator at 37 deg.C for 16 hr.

3. And (3) detecting cytokines:

③ diluting FITC-labeled anti-TNF- α monoclonal antibody MAb FITC-3C1 (prepared in example 12) with cross reaction of cattle and sheep to 0.5 μ g/mL by using a membrane breaking agent, standing at room temperature for 15min, washing cells by using PBS containing 1% BSA, centrifuging at 2000rpm at 4 ℃ for 10min, discarding the supernatant, and repeating twice;

The result is shown in figure 4, and it can be seen from figure 4 that when detecting bovine peripheral blood mononuclear cell samples, the proportion of bovine TNF- α cells secreted by the tuberculosis-positive cattle after being stimulated by CFP-10 and ESAT-6 fusion proteins is higher than that of bovine TNF- α cells secreted by the tuberculosis-negative cattle, and the proportion of goat TNF- α cells secreted by the tuberculosis-positive sheep after being stimulated by CFP-10 and ESAT-6 fusion proteins is higher than that of the goat TNF- α cells secreted by the tuberculosis-negative sheep.

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

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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 or a passage cell strain thereof is disclosed, wherein the preservation number of the hybridoma cell strain is CCTCC NO: C2019129.

2. an anti-TNF- α antibody is prepared from the hybridoma cell strain with CCTCC NO: C2019129 or its subcultured cell strain.

3. An anti-TNF- α antibody, the anti-TNF- α antibody comprises a heavy chain variable region and a light chain variable region, the CDR of the light chain variable region comprises CDR-L1 shown in SEQ ID No.1, CDR-L2 shown in SEQ ID No.2 and CDR-L3 shown in SEQ ID No.3, the CDR of the heavy chain variable region comprises CDR-H1 shown in SEQ ID No.6, CDR-H2 shown in SEQ ID No. 7and CDR-H3 shown in SEQ ID No. 8.

4. The anti-TNF- α antibody of claim 3, wherein the anti-TNF- α antibody is a monoclonal antibody;

and/or, 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);

preferably, the amino acid sequence of the light chain of the anti-TNF- α antibody comprises the amino acid sequence shown as SEQ ID No. 5;

and/or, 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);

preferably, the amino acid sequence of the heavy chain of the anti-TNF- α antibody comprises the amino acid sequence shown as SEQ ID No. 10.

5. An isolated polynucleotide encoding the heavy chain variable region and/or the light chain variable region and/or the full length amino acid of the anti-TNF- α antibody of any one of claims 3-4.

6. A construct comprising the isolated polynucleotide of claim 5.

7. An antibody expression system comprising the construct or genome of claim 6 having integrated therein an exogenous polynucleotide according to claim 5.

8. The method for producing an anti-TNF- α antibody according to any one of claims 2 to 4, comprising the steps of culturing an antibody expression system according to claim 7 to express said antibody, purifying and isolating said antibody;

9. Use of an anti-TNF- α antibody according to any one of claims 2-4 in the preparation of a TNF- α assay kit.

10. A test kit comprising an anti-TNF- α antibody of any one of claims 2-4.