CN114292334B - Anti-cotinine specific antibodies, plasmid vectors and methods - Google Patents

Anti-cotinine specific antibodies, plasmid vectors and methods Download PDF

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Publication number
CN114292334B
CN114292334B CN202111594778.6A CN202111594778A CN114292334B CN 114292334 B CN114292334 B CN 114292334B CN 202111594778 A CN202111594778 A CN 202111594778A CN 114292334 B CN114292334 B CN 114292334B
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variable region
nucleotide sequence
cotinine
heavy chain
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CN114292334A (en
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洪淑凡
朱伟
武妮妮
宋静
余卫
敖翔
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HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
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HANGZHOU XIANZHI BIOTECHNOLOGY CO Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract

The application discloses an anti-cotinine specific antibody, a plasmid vector and a method, which comprise the steps of immunizing a mouse by a COT-KLH conjugate, separating spleen lymphocytes of the mouse, separating B lymphocytes specifically combined with the protein by a flow cytometry, amplifying antibody heavy chain and light chain variable region sequences in the B lymphocytes by single cell PCR, constructing the obtained sequences into a complete mouse IgG antibody sequence expression vector, expressing the monoclonal antibody by transient HEK293F cells, purifying the monoclonal antibody, respectively marking colloidal gold particles, determining dominant monoclonal antibodies by a colloidal gold competition method, and having important significance for evaluating the environmental tobacco smoke exposure degree, smoking amount and the influence degree of smoking on human health of a subject.

Description

Anti-cotinine specific antibodies, plasmid vectors and methods
Technical Field
The application belongs to the technical field of bioengineering. In particular to an anti-cotinine specific antibody, a plasmid vector and a method.
Background
The use of tobacco causes millions of deaths each year worldwide, creating serious public health problems. Tobacco smoke contains thousands of chemical substances, more than sixty of which are determined or suspected to have carcinogenic effects on human bodies, and when the exposure and hazard of the carcinogens are evaluated, proper biomarkers are often required to evaluate the delayed intake of tobacco, the intake of the carcinogens, the health effect and the like. Nicotine, which is an addictive alkaloid existing in tobacco, is absorbed into the blood circulation through the cavity, throat, trachea and alveoli in a short time, wherein about 80% of nicotine is first metabolized into Cotinine (COT) by cytochrome oxidase 2A6 (CYP 2 A6) in the liver, is mainly present in the blood, and then is further metabolized, and finally these metabolites are discharged through urine and the like. Cotinine in urine is therefore the most effective biomarker for assessing the subject's environment for tobacco smoke exposure, amount of smoking, and the extent to which smoking affects human health.
Although methods such as liquid chromatography, high performance liquid chromatography, gas chromatography and high performance liquid chromatography combined mass spectrometry have been used for detection of cotinine, these methods require sample pretreatment, and are costly and time consuming. The immunodetection has the advantages of strong antibody and antigen specificity, high sensitivity, simple operation, large flux, low cost and the like. The immune detection technology represented by the colloidal gold immune chromatography test paper detection method is particularly outstanding in the aspect of rapid diagnosis of cotinine, not only simplifies the detection step, but also improves the specificity and sensitivity of cotinine detection.
Therefore, the preparation of cotinine monoclonal antibodies becomes a main mode of cotinine specific detection and diagnosis.
Disclosure of Invention
In order to achieve the above design objective. In a first aspect of the application, there is provided an anti-cotinine specific antibody comprising a light chain and a heavy chain, wherein the variable region of the light chain has the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.
Further, the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.1 is shown as SEQ ID NO. 5; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.2 is shown as SEQ ID NO. 6.
In a second aspect of the present application, there is provided a plasmid vector comprising the light chain variable region nucleotide sequence shown as SEQ ID NO. 5.
In a third aspect of the present application, there is provided a plasmid vector comprising a heavy chain variable region nucleotide sequence as set forth in SEQ ID NO. 6.
In a fourth aspect of the application, there is provided an anti-cotinine specific antibody comprising a light chain and a heavy chain, wherein the variable region of the light chain has an amino acid sequence shown in SEQ ID NO. 3; the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO. 4.
Further, the nucleotide sequence of the variable region amino acid sequence of the light chain shown as SEQ ID NO.3 is shown as SEQ ID NO. 7; the nucleotide sequence of the variable region amino acid sequence of the heavy chain shown as SEQ ID NO.4 is shown as SEQ ID NO. 8.
In a fifth aspect of the present application, there is provided a plasmid vector comprising the light chain variable region nucleotide sequence shown as SEQ ID NO. 7.
In a sixth aspect of the present application, there is provided a plasmid vector comprising a heavy chain variable region nucleotide sequence as set forth in SEQ ID NO. 8.
In a seventh aspect of the present application, there is provided a method for eukaryotic expression of an antibody against cotinine specificity, comprising the steps of:
a) The nucleotide sequence of the light chain variable region and the nucleotide sequence of the heavy chain variable region are respectively bridged with the nucleotide sequence of the mouse IgG light chain constant region and the nucleotide sequence of the heavy chain constant region by PCR, and then are respectively connected with plasmid vectors to construct eukaryotic cell expression vectors;
b) Transfecting the eukaryotic expression vector in the step a) to HEK293F cells to express to obtain anti-Cotinine (COT) monoclonal antibodies;
c) Purifying monoclonal antibodies, respectively marking colloidal gold particles, and determining dominant monoclonal antibodies by a colloidal gold competition method;
the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.5 or as SEQ ID NO. 7;
the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.6 or SEQ ID NO. 8.
The application has the advantages that: an anti-cocaine specific antibody is provided.
Detailed Description
Although the following embodiments describe the design concept of the present application in more detail, these descriptions are merely descriptions of the design concept of the present application, and not limitations of the design concept of the present application, and any combination, addition or modification of the design concept of the present application will fall within the scope of the present application.
Cotinine (COT) monoclonal antibody preparation
COT-KLH immunogen was subcutaneously injected at multiple sites on the dorsum of the 4-6 week old Balb/c female mice, and 100. Mu.g of COT-KLH was emulsified with Freund's complete adjuvant at the first immunization, 400. Mu.l each. A second boost was performed 20 days later, and 80. Mu.g of COT-KLH was emulsified with Freund's incomplete adjuvant, 400. Mu.l/min. A third boost was performed 15 days later and the procedure was the same as the second boost. After 20 days, 120. Mu.g of COT-KLH was injected intraperitoneally and the mice were sacrificed after 72 hours, spleens were removed and mouse spleen lymphocytes were isolated with a mouse lymphocyte separation kit (Tianjin ocean Biometrics Co., ltd.). And (3) adding a fluorescent marker probe prepared by COT-KLH to dye target B lymphocytes, and separating single B cells capable of expressing specific antibodies from the target B lymphocytes by using a flow cytometry fluorescence sorting technology. mRNA of single B cell is extracted, RT-PCR is performed to synthesize cDNA, cDNA is taken as a template, universal degenerate primers of a murine antibody variable region are used for respectively amplifying nucleotide sequences for coding an antibody light and heavy chain variable region, and the nucleotide sequences are respectively connected with a constant region and then inserted into a pcDNA3.1 (+) vector to construct recombinant plasmid capable of expressing specific antibody light and heavy chains. Light chain plasmid and heavy chain plasmid of the same antibody were mixed according to 1:1 mass ratio, and then transfected into HEK293F cells for culture, 5% CO 2 After shaking culture at constant temperature of 37℃for 7 days at 120rpm, the cell culture broth was collected and purified by Protein A affinity chromatography to obtain monoclonal antibodies. Purity was measured by coomassie blue staining after 10% sds-PAGE electrophoresis. And meanwhile, ELISA screening is carried out on the purified monoclonal antibody, and the screening steps are as follows:
coating: diluting COT-KLH and KLH with coating solution to a final concentration of 1. Mu.g/mL, adding an ELISA plate (Shenzhen Jin Canhua Co., ltd.) at a final concentration of 100. Mu.L/well, and washing with a washing solution 1 time with a DEM-3 plate washer (Zhongshan university Daan Gene Co., ltd.) after overnight at 4deg.C;
closing: adding a sealing liquid at 200 mu L/hole, sealing for 2 hours at 37 ℃, and washing 1 time by using a washing liquid through a plate washer;
sample adding: adding purified monoclonal antibody and control serum diluted in a certain proportion into coating holes of COT-KLH conjugate and KLH respectively, incubating for 1h at 37 ℃ with 100 mu L/hole, and washing 3 times by using a washing liquid of a plate washer;
adding enzyme-labeled antibody: adding fresh diluted HRP enzyme-labeled secondary antibody into COT-KLH conjugate and KLH coated holes at a rate of 100 mu L/hole, incubating at 37 ℃ for 30 minutes, and washing 4 times by using a washing liquid through a plate washer;
adding a color development liquid: adding 50 mu L of each of the developing solution A and the developing solution B into each hole, and developing for 10 minutes at 37 ℃ in a dark place;
terminating the reaction: adding a stop solution at a concentration of 50. Mu.L/well;
and (3) result judgment: and on an enzyme labeling instrument, at the wavelength of 450nm, reading absorbance values after blank holes are zeroed. The serum of immunized mice was used as a positive control. The results showed that 5 positive clones had higher absorbance values and no color development in KLH wells, and 5 sequences were obtained by sequencing, 1A10,8D5,7G9,2E6,3D3 each.
The relevant solution formulation is as follows:
coating liquid: na (Na) 2 CO 3 1.5g，NaHCO 3 2.9g, add ddH 2 O was fixed to 1000mL (pH 9.6).
Sealing liquid: na (Na) 2 HPO 4 .12H 2 O 2.68g，NaH 2 PO 4 .2H 2 O0.39 g, naCl 8.5g, bovine serum albumin 20g, ddH 2 O was fixed to 1000mL (pH 7.4).
Washing liquid: na (Na) 2 HPO 4 .12H 2 O 2.68g，NaH 2 PO 4 .2H 2 O0.39g,NaCl 8.5g,Tween-20.5 mL, add ddH 2 O was fixed to 1000mL (pH 7.4).
Color development liquid A:200mg TMB is dissolved in 100mL absolute ethanol, add ddH 2 O is fixed to 1000mL.
Color development liquid B: citric acid 2.1g, na 2 HPO 4 .12H 2 O71 g, add ddH 2 O is fixed to 1000mL.
When in use, the utility model is characterized in that: 1mL of color developing solution A+1mL of color developing solution B+0.4. Mu.L of 30% H 2 O 2
Stop solution: 2M H 2 SO 4 21.7mL of concentrated H 2 SO 4 Adding ddH 2 O is fixed to 1000mL.
Preparation of colloidal gold pad
10ml of 0.01% colloidal gold solution is taken, 20 mu L of 0.2mol/L potassium carbonate solution is added, 100 mu g of monoclonal antibody is added after fully and uniformly mixing, 1ml of 10% BSA (bovine serum albumin) solution is added for sealing after standing for 2 hours at room temperature, centrifugation (10000 rpm/min and 25 min) is carried out after sealing treatment for 2 hours, and after supernatant is discarded, 1ml of complex solution is used for fully dissolving sediment. The dissolved gold solution was uniformly sprayed on a 6mm wide glass fiber with a gold spraying and film drawing instrument (Sangzhou Houzan technologies Co., ltd.) at a concentration of 6. Mu.l/cm, and then was dried by air blowing at 37℃in an electric heating air drying oven (Shanghai-Heng science instruments Co., ltd.) for 1 hour.
The relevant solution formulation is as follows:
0.01% colloidal gold solution: 1ml of 1% chloroauric acid solution and 1.4ml of 1% citric acid solution, and adding ultrapure water to heat and dissolve the mixture, and fixing the volume to 100ml.
1% chloroauric acid solution: auCL (AuCL) 3 .HCl.4H 2 O powder 1g was dissolved in ultrapure water and the volume was fixed to 100ml.
1% citric acid solution: 1g of citric acid crystal was dissolved in ultrapure water and the volume was fixed to 100ml.
0.2mol/L potassium carbonate solution: 27.64 g of potassium carbonate was dissolved in ultrapure water and the volume was set to 1000ml.
And (3) a complex solution: tris base 6.057g was dissolved in 800ml of ultrapure water, the pH was adjusted to 8.0 with an appropriate amount of HCL, and the volume was adjusted to 1000ml by adding ultrapure water.
Preparation of nitrocellulose Membrane (NC Membrane)
COT-KLH antigen was diluted with the coating solution (final concentration: 1 mg/ml), and then uniformly coated on a nitrocellulose membrane (Sartorius) at 1. Mu.l/cm by a gold spraying and film drawing machine (Sanguson technologies Co., ltd.), which was T line. Sheep anti-mouse solution (final concentration 1 mg/ml) was uniformly coated on nitrocellulose membrane at 1. Mu.l/cm by a metal spraying and film drawing instrument (Hangzhou NZan technology Co., ltd.), which is line C. After the film coating was completed, the nitrocellulose film was dried at 37℃for 12 hours in an electrothermal forced air drying oven (Shanghai-Heng scientific instruments Co., ltd.).
Preparation of colloidal gold immunity detection card
Assembling a test strip: sequentially overlapping and pasting on a PVC bottom plate: (1) Spraying NC film with COT-KLH antigen as detection region and goat anti-mouse IgG as quality control region; (2) Gold pads coated with colloidal gold-labeled anti-Cotinine (COT) monoclonal antibodies (1A10,8D5,7G9,2E6,3D3); (3) The sample pad is a glass fiber membrane treated by 2% Tween-20; (4) And (3) cutting the water absorbing paper into a width of 4mm after the assembly is completed, mounting a reagent card strip shell, and compacting to obtain the colloidal gold immunochromatography detection card.
Monoclonal antibody screening
The urine samples of smokers and non-smokers were collected, loaded at 80. Mu.L/well, and after 15min at room temperature, read through a colloidal gold colorimetric card, and the results are detailed in Table 1.
TABLE 1 colloidal gold colorimetric card value statistics
From the above table, 2E6 and 8D5 labeled monoclonal antibodies were two dominant monoclonal antibodies for detecting COT.
In conclusion, the application uses COT-KLH conjugate, B lymphocyte combined with the protein specifically is separated by a flow cytometry, the antibody heavy chain and light chain variable region sequences in the B lymphocyte are amplified by single cell PCR, and the obtained sequences are constructed into a complete mouse IgG antibody sequence expression vector to express anti-Cotinine (COT) monoclonal antibody, so that the application has important significance in evaluating the tobacco smoke exposure degree, smoking amount and smoking influence degree on human health of the environment of a subject.
The design purpose is as follows: to improve the shortcomings of the traditional monoclonal antibody preparation, a flow cytometer is adopted to screen out the lymphocyte B cells which are specifically combined with Cotinine (COT), and the nucleotide sequence of the antibody is amplified, and then the monoclonal antibody is prepared through transient expression. Compared with the traditional monoclonal antibody preparation, the preparation and use time is shorter, the stability of the obtained monoclonal antibody is higher, the uniformity is better, and the batch-to-batch difference is greatly reduced.
The application comprises the following steps: (1) The COT-KLH conjugate is used for immunizing Balb/c mice for multiple times, spleen is taken out, spleen lymphocytes are separated by using a lymphocyte separation liquid kit, B lymphocyte suspension is separated by using a BD FACS flow cytometry, the B lymphocyte suspension is collected in a 96-well PCR plate containing proper cell lysate, an RNase inhibitor and a PCR reaction reagent, a mixture of forward primers is designed for different leader sequences of a heavy chain and light chain variable region of an antibody, reverse primers are specifically complementary to an antibody constant region, mRNA is reverse transcribed into cDNA, a corresponding antibody variable region nucleotide sequence is cloned by RT-PCR, gel electrophoresis analysis, purification and sequencing are carried out, and finally the antibody variable region nucleotide sequence capable of combining with protein is obtained. (2) The heavy and light chain variable region sequences were constructed into complete murine IgG expression vectors and monoclonal antibodies were expressed using HEK293F cells, purified using Protein a affinity chromatography, and colloidal gold particles were labeled, respectively. (3) The colloidal gold immunochromatography screening platform is utilized to display that the 2E6 and 8D5 colloidal gold labeled monoclonal antibodies are dominant monoclonal antibodies for detecting cotinine.
SEQ ID NO1: anti-Cotinine (COT) specific antibody 2E6 light chain variable region amino acid sequence;
SEQ ID NO2: anti-Cotinine (COT) specific antibody 2E6 heavy chain variable region amino acid sequence;
SEQ ID NO3: an anti-Cotinine (COT) specific antibody 8D5 light chain variable region amino acid sequence;
SEQ ID NO4: anti-Cotinine (COT) specific antibody 8D5 heavy chain variable region amino acid sequence;
SEQ ID NO5: anti-Cotinine (COT) specific antibody 2E6 light chain variable region nucleotide sequence;
SEQ ID NO6: anti-Cotinine (COT) specific antibody 2E6 heavy chain variable region nucleotide sequence;
SEQ ID NO7: anti-Cotinine (COT) specific antibody 8D5 light chain variable region nucleotide sequence;
SEQ ID NO8: anti-Cotinine (COT) specific antibody 8D5 heavy chain variable region nucleotide sequence.
Sequence listing
<110> Hangzhou xian to Biotechnology Co., ltd
<120> anti-cotinine specific antibodies, plasmid vectors and methods
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Claims (4)

1. An anti-cotinine specific antibody comprising a light chain and a heavy chain, characterized in that:
the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO. 1;
the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO. 2.
2. An anti-cotinine specific antibody according to claim 1, wherein:
the nucleotide sequence of the amino acid sequence of the variable region of the light chain shown as SEQ ID NO.1 is shown as SEQ ID NO. 5;
the nucleotide sequence of the amino acid sequence of the variable region of the heavy chain shown as SEQ ID NO.2 is shown as SEQ ID NO. 6.
3. A plasmid vector combination, characterized in that: the plasmid vector combination contains nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6.
4. A method of preparing an anti-cotinine specific antibody as claimed in claim 1 or 2, comprising the steps of:
a) The variable region nucleotide sequence of the light chain and the variable region nucleotide sequence of the heavy chain are respectively bridged with the mouse IgG light chain constant region nucleotide sequence and the heavy chain constant region nucleotide sequence by PCR, and then are respectively connected with plasmid vectors to construct eukaryotic cell expression vectors;
b) Transfecting the eukaryotic expression vector in the step a) to HEK293F cells to express to obtain anti-cotinine monoclonal antibodies;
c) Purifying the monoclonal antibody, and determining dominant monoclonal antibody through a colloidal gold-colloidal gold competition method experiment;
the nucleotide sequence of the variable region of the light chain is shown as SEQ ID NO. 5;
the nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO. 6.
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