CN108795878B

CN108795878B - Hybridoma cell strain secreting anti-CST 1 monoclonal antibody, monoclonal antibody and application thereof

Info

Publication number: CN108795878B
Application number: CN201810508793.6A
Authority: CN
Inventors: 孙玉龙; 王力军; 杨亚云; 何林富; 王�义
Original assignee: Shanghai Liangrun Biomedical Technology Co ltd
Current assignee: Shanghai Liangrun Biomedical Technology Co ltd
Priority date: 2018-05-24
Filing date: 2018-05-24
Publication date: 2022-01-11
Anticipated expiration: 2038-05-24
Also published as: CN108795878A

Abstract

The invention relates to a hybridoma cell strain secreting anti-CST 1 monoclonal antibody, monoclonal antibody and application thereof, comprising hybridoma cell strains 4G2 and 4E7, wherein the hybridoma cell strains are preserved in China general microbiological culture Collection center in 2017, 12 months and 28 days, and the preservation numbers are CGMCC NO.15199 and CGMCC NO.15198 respectively; the invention also provides an anti-CST 1 monoclonal antibody secreted by the hybridoma cell strain; the two monoclonal antibodies provided by the invention can be used for detecting CST1 protein in a sample, and have strong specificity and high sensitivity; the two monoclonal antibodies specifically recognize different segments of CST1, can detect the content of human cystatin SN CST1 in body fluid and tissues through pairing, and can be applied to the detection of CST1 protein overexpression or the diagnosis of products of diseases characterized by CST1 protein abnormal expression.

Description

Hybridoma cell strain secreting anti-CST 1 monoclonal antibody, monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a hybridoma cell strain secreting a CST 1-resistant monoclonal antibody, a monoclonal antibody secreted by the hybridoma cell strain and application of the monoclonal antibody.

Background

Cystatin SN (CST1) is a member of the human cystatin family, a protein of 141 amino acids encoded by the CST1 gene, with a molecular weight of 16.4 Kda. CST1 contains two disulfide bonds in its molecule, and is a typical secreted protein distributed in body fluids and secretions, such as tears, saliva, serum, plasma, and the like.

With the continuous and intensive research on the pathogenesis of tumors, CST1 is found to be one of the members of the endogenous inhibitor family of cysteine proteases as cathepsin, and plays a very important role in the processes of tumor generation, development, infiltration and metastasis.

The research results show that the expression level of the Cystatin family members in different tumors is increased, for example, the expression level of Cystatin C in ovarian cancer and head and neck cancer is increased, and the expression level of Cystatin A in non-small cell lung cancer is increased. CST1 has been reported to be capable of abnormal expression in esophageal, lung, and breast cancers. The quantitative detection of CST1 is of great clinical significance. Polyclonal antibodies are mostly adopted in the existing CST1 detection system; the specificity of the antibody is poor, the highly homologous family proteins of CST1 cannot be distinguished, and the clinical utility of the antibody on CST1 detection is limited due to the titer or specificity of the antibody, so that the specificity of sample detection is reduced, and the sensitivity is influenced to a certain extent. Therefore, a monoclonal antibody for specifically recognizing the CST1 protein is urgently needed to be obtained, the detection requirement of clinical samples is met, and the detection sensitivity and specificity are improved.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a hybridoma cell line secreting anti-CST 1 monoclonal antibody; the second purpose of the invention is to provide a monoclonal antibody secreted by a hybridoma cell strain secreting the anti-CST 1 monoclonal antibody; the third purpose of the invention is to provide a kit containing the monoclonal antibody; the fourth purpose of the invention is to provide the application of the monoclonal antibody in preparing a reagent for detecting CST1 protein.

In order to achieve the purpose, the invention provides the following technical scheme:

1. hybridoma cell strain secreting monoclonal antibody against CST1 comprises hybridoma cell strains 4G2 and 4E7, and is preserved in Beijing China general microbiological culture Collection center with preservation numbers of CGMCC NO.15199 and CGMCC NO.15198 respectively.

2. The monoclonal antibody secreted by the anti-CST 1 monoclonal antibody hybridoma cell strain is disclosed.

Preferably, the epitope of the monoclonal antibody is at the C-terminal of CST1 protein or M segment of CST1 protein, and the amino acid sequence at the C-terminal of CST1 protein is shown in SEQ ID NO. 5; the amino acid sequence of the M segment in the CST1 protein is shown as SEQ ID NO. 4.

3. A kit containing the monoclonal antibody.

Preferably, the kit comprises an enzyme label plate containing the monoclonal antibody, a calibrator CST1 protein, a detection antibody marked by horseradish peroxidase, a chromogenic substrate and a stop solution.

More preferably, the chromogenic substrate is TMB, the stop solution is 2M sulfuric acid, the coating antibody is a monoclonal antibody secreted by hybridoma cell line 4G2, and the detection antibody is a monoclonal antibody secreted by hybridoma cell line 4E 7.

4. The monoclonal antibody is applied to preparation of a reagent for detecting CST1 protein.

Preferably, the reagent for detecting the CST1 protein is a reagent for detecting the excessive expression of the CST1 protein or diagnosing the abnormal expression of the CST1 protein.

Preferably, the capture antibody of the CST1 protein detection reagent is a monoclonal antibody secreted by a hybridoma cell line 4G2, and the detection antibody is a monoclonal antibody secreted by a hybridoma cell line 4E 7.

The invention has the beneficial effects that: according to the invention, 2 monoclonal antibodies aiming at different epitopes of CST1 are obtained by obtaining a hybridoma cell strain secreting the anti-CST 1 monoclonal antibody, the specificity is strong, the sensitivity is high, the content of a human cysteine protease inhibitor SN CST1 in body fluid and tissues can be detected by matching, and the monoclonal antibody can be applied to the application of detecting the over-expression of CST1 protein or diagnosing products of diseases characterized by the abnormal expression of CST1 protein.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 is an SDS-PAGE electrophoresis of recombinant CST1 protein.

FIG. 2 is an SDS-PAGE electrophoresis of 4G2 and 4E7 antibodies.

FIG. 3 shows the detection result of CST1 truncated Western blot WB.

FIG. 4 is a CST1 test kit calibration curve wherein the Y-axis represents OD values and the X-axis represents the concentration of CST1 calibrator.

Biological preservation

The hybridoma cell strains 4G2 and 4E7 are stored in the common microorganism center of China Committee for culture Collection of microorganisms at 28.12.2017, the storage address is the microorganism research institute of China academy of sciences No.3 of Western Lu 1 of North Chen, south China, Beijing, the market, the preservation number of hybridoma cell strain 4G2 is CGMCC No.15199, the preservation number of hybridoma cell strain 4E7 is CGMCC No.15198, and the hybridoma cell strains are classified and named as hybridoma cell strain 4G2 and hybridoma cell strain 4E 7.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers.

Example 1 recombinant protein expression and purification of CST1

Extracting RNA from esophageal cancer tissues, then using cDNA obtained by random primer RT-PCR as a template, designing a primer for cloning CST1 gene, wherein the primer specifically comprises the following components: CST 1-F: 5' -cccaagcttgccaccatggcccagtatctgagtacc-3’(SEQ ID NO.1)；CST1-R：5’-ggattcttgacacctggatttcac-3’)(SEQ ID NO.2)。

The amplified CST1 gene was inserted into pcDNA3.1 vector containing 6 XHis tag to obtain CST 1-pcDNA3.1. Then CST1-pc DNA3.1 is transformed into DH5 alpha, after positive clone is picked up and mass culture, recombinant plasmid CST1-pcDNA3.1 is extracted by a high-purity plasmid extraction kit. The recombinant plasmid is transferred into 293T cell, and pcDNA3.1 empty vector is simultaneously transfected as negative control, respectively in DMEM culture medium containing 10% fetal calf serum at 37 ℃ and 5% CO₂After culturing for 72 hours under the conditions, the supernatant was collected and filtered through a 0.22 μm filter.

The 500mL filtrate was subjected to Ni-NTA affinity chromatography under non-denaturing conditions in 50mM PBS, 10mM imidazole, 150mM NaCl, pH7.6 as equilibration buffer. After the sample loading is finished, washing 10 mL; eluted with 50mM PBS 250mM imidazole, 150mM NaCl, pH7.6 and the eluate was collected. The protein solution was concentrated using a 3kD ultrafiltration tube and the protein was stored in PBS buffer at pH 7.450mM and at-80 ℃. The purity of the purified protein is identified by SDS-PAGE electrophoresis, the molecular weight is about 15kD, and the grey analysis shows that the purity of the protein reaches more than 95 percent, which is shown in figure 1.

Example 2 preparation of CST1 monoclonal antibody hybridoma cell line

Mouse immunization: 3 BALB/c mice were immunized 3 times with CST1 protein, 3 at a time. Each mouse was immunized with 50. mu.g of antigen per time. Mixing the first immunization CST1 antigen with Freund's complete adjuvant 1:1, then mixing CST1 with Freund's incomplete complete adjuvant 1:1, immunizing once every two weeks for 3 times, cutting off the tail and collecting blood after ten days of the third immunization, and detecting the serum titer.

Hybridoma cell fusion: detecting the serum titer of a mouse to reach 1:100000, performing intraperitoneal boosting immunization (30 mu g) by using CST1 antigen (without adjuvant), aseptically taking splenocytes of the immunized mouse and SP2/0 mouse myeloma cells to mix in a 50mL centrifuge tube according to a ratio of 4:1 after 3 days, washing twice by using a culture medium, discarding supernatant, adding 1mL preheated 50% PEG-1450 for acting for 1min, slowly shaking for 90s in a water bath, immediately and slowly dripping 15mL of serum-free DMEM culture medium preheated at 37 ℃, carrying out the water bath at 37 ℃ for 5min, then supplementing the serum-free DMEM culture medium to 40mL, centrifuging at 1000rpm/mim for 10min, discarding supernatant, adding 40mL of preheated HAT culture medium, slightly blowing, uniformly mixing, transferring to a 96-hole culture plate with the laid cells, and placing 100 mu L of each hole in a culture box for culture. And (3) identifying cell supernatants, and carrying out cloning screening on the positive hybridomas for 3 times to obtain hybridoma cell 4 strains secreting specific monoclonal antibodies, wherein the hybridoma cell 4 strains are respectively 4G2, 4E7 and 4F 7.

Example 3 CST1 monoclonal antibody preparation, purification, potency detection and specificity analysis

Preparing monoclonal antibody ascites: culturing 4 selected hybridoma cell strains in culture medium to 90% density, collecting cells, diluting to 3 × 10⁶one/mL. After 10-12 weeks BALB/c mice are adaptively raised for one week, the mice are intraperitoneally injected with immunosuppressant liquid paraffin, 0.5 mL/mouse, and 7d later, the abdomen is inoculated with hybridoma cells with stable secretion of antibody and better state, about (1-2) -10%⁶After 7-10 days, after the abdomen of the mouse is expanded, the ascites is extracted, and the abdominal water supernatant is collected.

Antibody purification: ascites from mice was collected, the solution was filtered through a 0.45 μm filter, ammonium sulfate solid was added to a final concentration of 50%, and after stirring well at 4 ℃, the precipitate was collected by standing at 12000rpm and re-dissolved in 50mM PBS. The resulting crude purified antibody was applied to a Protein A-Sepharose affinity column at a flow rate of 1mL/min, washed with 5 column volumes of binding buffer (50mMPBS, pH7.0), and then the antibody was eluted with 0.1M glycine-hydrochloric acid solution, pH2.7 (1M Tris buffer pH 9.0 was added to each collection tube in advance for neutralization) to give the objective antibody, and dialyzed 3 times against 50mM PBS solution. The dialyzed antibodies were subjected to 10% SDS-PAGE, and the results showed that 3 antibodies had bands at 55kD and 25kD, and had greater than 90% purity in grayscale analysis, and the SDS-PAGE of 4G2 and 4E7 antibodies is shown in FIG. 2.

Detecting the titer of the monoclonal antibody: the microplate was coated with 1. mu.g/ml of recombinant CST1 protein in carbonate buffer (pH 9.5), 100. mu.l volume at 4 ℃ overnight, each antibody was diluted in a gradient (1: 1000, 1: 2000, 1: 4000, 1: 64000, 1:128000), goat anti-mouse IgG-HRP (50ng/ml) was added, and the purified monoclonal antibody titers (S/N >2.1), 4G2, 4E7 and 4F7 were all 1: 128000.

monoclonal antibody specificity analysis: microplates were coated overnight at 4 ℃ in a volume of 100. mu.l CBS buffer containing 1. mu.g/ml of CST1, CST4 and CST3 proteins, respectively, and each antibody was diluted in multiples (500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.25ng/ml, 15.625ng/ml, 7.8125ng/ml and 0ng/ml) and then added to microwell reaction plates followed by goat anti-mouse IgG-HRP (50ng/ml), with the results shown in Table 1. The results confirmed that 4G2 specifically recognizes CST1 protein, and 4E7 and 4F7 react with both CST1 and CST4 protein, so 4G2 can be used as a capture antibody of the kit for specifically recognizing CST 1.

TABLE 1 analysis of monoclonal antibody specificity

Example 4, validation of monoclonal antibody pairing by CST1

The microwell reaction plate was coated with 3. mu.g/mL 4G2 antibody, 100. mu.l of CST1 calibrator was added at different concentrations (6.25-1600U/mL), incubated at 37 ℃ for 60min, after washing, 100. mu.L of HPR-labeled 4E7 antibody and 4F7 antibody at 100ng/mL were added, and incubated at 37 ℃ for 60min, and after washing, the substrate was added and absorbance was measured in a microplate reader, with the results shown in Table 2. From the results, 4G2 was better paired with 4E7, and the sensitivity and absorbance were higher.

TABLE 2 results of pairing of the CST1 monoclonal antibody

Calibrator U/mL	4E7	4F7
				0	0.054	0.059
50	0.155	0.057
			100	0.257	0.062
200	0.457	0.124
			400	0.802	0.233
800	1.305	0.453
			1600	1.963	0.819

Example 5, in-situ characterization of CST1 monoclonal antibody

Recombinant truncated CST1 protein construction expression: according to the protein sequence of CST1, a sequence at the N, M, C end and a full-length protein of CST1 (marked as CST1-F) are designed, an expression vector pET30a is connected, and the protein is transferred into an expression strain BL21(DE 3). CST1 was induced overnight in LB medium at 25 ℃ with IPTG 0.1mmol/L to intercept recombinant protein soluble expression. Prokaryotic recombinant expression is subjected to affinity purification by 6 × His tags, and the purified protein is subjected to SDS-PAGE electrophoresis, and the purity is over 90 percent.

Fragment CST-N (21-47): WSPKEEDRIIPGGIYNADLNDEWVQRA (SEQ ID NO. 3);

fragment CST-M (48-93): LHFAISEYNKATKDDYYRRPLRVLRARQQTVGGVNYFFDVEVGRTI (SEQ ID NO. 4);

CST-C fragment (112-141): LQKKQLCSFEIYEVPWENRRSLVKSRCQES (SEQ ID NO.5)

Fragment CST-F (21-141): WSPKEEDRIIPGGIYNADLNDEWVQRALHFAISEYNKATKDDYYRRPLRVLRARQQTVGGVNYFFDVEVGRTICTKSQPNLDTCAFHEQPELQKKQLCSFEIYEVPWENRRSLVKSRCQES (SEQ ID NO.6)

Western blotting WB confirmed antibody recognition epitope: recombinant protein CST1-N/M/C, CST1 full-length 4 proteins were sampled at 5. mu.g and subjected to 15% SDS-PAGE. After the electrophoresis was completed, the protein was transferred to 0.45pg mVDF under a constant pressure of 150V. After the membrane transfer was completed, blocking was performed for two hours at 37 ℃ using PBST solution containing 5% BSA. Each was incubated with 5. mu.g/mL antibody (4G2/4E7) solution for 1 hour at 37 ℃. After the incubation was complete, 50ng/ml rabbit anti-mouse IgG-HRP was added and incubated for 1 hour. And after the cleaning is finished, adding DAB color developing solution for color development. The full-length protein of CST1 served as a positive control, and brown precipitated material at the position of CST1-N/M/C fragment, indicating that the antibody recognizes the fragment of interest. The results showed that the 4G2 antibody recognized the C segment of CST1 protein and the 4E7 antibody recognized the M segment of CST1 protein, as shown in fig. 3.

Example 6 CST1 detection kit

And (3) drawing a calibration curve: first, capture antibody 4G2 was coated overnight at 4 ℃ on an enzyme plate at a concentration of 2.5. mu.g/mL, recombinant human CST1 calibrator protein was diluted with 20% FBS solution to 0U/mL, 20U/mL, 40U/mL, 80U/mL, 160U/mL, 320U/mL, 640U/mL at 100. mu.L per well, and horseradish peroxide-labeled 4E7 antibody was added at a concentration of 20ng/mL at 100. mu.L per well and incubated at 37 ℃ for 1 hour. The reaction was stopped by washing 3 times with PBST, developing the substrate with TMB for 15min and adding 2M sulfuric acid solution, and the OD450 read. The CST1 content of the tested sample was calculated from the calibration curve. The linear range of the calibration curve is 20-640U/mL, and FIG. 4 is a calibration curve of the CST1 detection kit, wherein the Y axis represents OD value pairs, and the X axis represents concentration pairs of CST1 calibrators.

The CST1 detection kit is used for esophageal cancer diagnosis, 100 pre-operation sera of esophageal cancer patients are collected from hospitals; 245 healthy blood donor sera were collected from the blood station at the same time. The kit is used for detecting the concentration of CST1 in esophageal cancer and serum of normal human by using CST 1. The ROC curve statistical result shows that the area under the curve is 0.87, 101U/mL is used as a detection reference value, the specificity of the CST1 detection kit is 86%, and the sensitivity is 79%.

In conclusion, the 2 monoclonal antibodies provided by the invention can be used for detecting the CST1 protein in the sample, and have strong specificity and high sensitivity. The 2 monoclonal antibodies specifically recognize different segments of CST1, can detect the content of human cystatin SN CST1 in body fluid and tissues through matching, and can be applied to the detection of CST1 protein over-expression or the diagnosis of products of diseases characterized by CST1 protein abnormal expression.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shanghaihuang biomedical science and technology Limited

<120> secretion anti-CST 1 monoclonal antibody hybridoma cell strain, monoclonal antibody and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cccaagcttg ccaccatggc ccagtatctg agtacc 36

<210> 2

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggattcttga cacctggatt tcac 24

<210> 3

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Trp Ser Pro Lys Glu Glu Asp Arg Ile Ile Pro Gly Gly Ile Tyr Asn

1 5 10 15

Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Ala

20 25

<210> 4

<211> 46

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Leu His Phe Ala Ile Ser Glu Tyr Asn Lys Ala Thr Lys Asp Asp Tyr

1 5 10 15

Tyr Arg Arg Pro Leu Arg Val Leu Arg Ala Arg Gln Gln Thr Val Gly

20 25 30

Gly Val Asn Tyr Phe Phe Asp Val Glu Val Gly Arg Thr Ile

35 40 45

<210> 5

<211> 30

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Leu Gln Lys Lys Gln Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Trp

1 5 10 15

Glu Asn Arg Arg Ser Leu Val Lys Ser Arg Cys Gln Glu Ser

20 25 30

<210> 6

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Trp Ser Pro Lys Glu Glu Asp Arg Ile Ile Pro Gly Gly Ile Tyr Asn

1 5 10 15

Ala Asp Leu Asn Asp Glu Trp Val Gln Arg Ala Leu His Phe Ala Ile

20 25 30

Ser Glu Tyr Asn Lys Ala Thr Lys Asp Asp Tyr Tyr Arg Arg Pro Leu

35 40 45

Arg Val Leu Arg Ala Arg Gln Gln Thr Val Gly Gly Val Asn Tyr Phe

50 55 60

Phe Asp Val Glu Val Gly Arg Thr Ile Cys Thr Lys Ser Gln Pro Asn

65 70 75 80

Leu Asp Thr Cys Ala Phe His Glu Gln Pro Glu Leu Gln Lys Lys Gln

85 90 95

Leu Cys Ser Phe Glu Ile Tyr Glu Val Pro Trp Glu Asn Arg Arg Ser

100 105 110

Leu Val Lys Ser Arg Cys Gln Glu Ser

115 120

Claims

1. The hybridoma cell strain secreting the anti-CST 1 monoclonal antibody is characterized in that: the hybridoma cell strain secreting the anti-CST 1 monoclonal antibody consists of hybridoma cell strains 4G2 and 4E7, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation numbers of CGMCC NO.15199 and CGMCC NO.15198 respectively.

2. The monoclonal antibody secreted by the anti-CST 1 monoclonal antibody hybridoma cell line of claim 1.

3. The monoclonal antibody of claim 2, characterized in that: the epitope of the monoclonal antibody is positioned in the C segment of CST1 protein recognized by the 4G2 antibody, and the amino acid sequence positioned at the C end of the CST1 protein is shown as SEQ ID NO. 5; the epitope of the monoclonal antibody is positioned in the M segment of CST1 protein recognized by the 4E7 antibody, and the amino acid sequence of the M segment of the CST1 protein is shown as SEQ ID NO. 4.

4. A kit comprising the monoclonal antibody of claim 2 or 3.

5. The kit of claim 4, wherein: the kit comprises an ELISA plate coated with the monoclonal antibody of claim 2 or 3, a calibrator CST1 protein, a detection antibody labeled by horseradish peroxidase, a chromogenic substrate and a stop solution.

6. The kit of claim 5, wherein: the chromogenic substrate is TMB, the stop solution is 2M sulfuric acid, the coating antibody is a monoclonal antibody secreted by a hybridoma cell strain 4G2, and the detection antibody is a monoclonal antibody secreted by a hybridoma cell strain 4E 7.

7. Use of the monoclonal antibody of claim 2 or 3 for the preparation of a reagent for the detection of CST1 protein.

8. Use according to claim 7, characterized in that: the reagent for detecting the CST1 protein is a reagent for detecting the excessive expression of the CST1 protein or diagnosing the abnormal expression of the CST1 protein.

9. Use according to claim 7, characterized in that: the capture antibody of the CST1 protein detection reagent is a monoclonal antibody secreted by a hybridoma cell line 4G2, and the detection antibody is a monoclonal antibody secreted by a hybridoma cell line 4E 7.