阅读说明：本技术 抗hpv16e7蛋白单抗69e2、杂交瘤细胞株及其制备方法和应用 (Monoclonal antibody 69E2 for resisting HPV16E7 protein, hybridoma cell strain, and preparation method and application thereof ) 是由 胡仁建 蔡家利 于 2020-03-16 设计创作，主要内容包括：本发明公开了抗HPV16E7蛋白单抗69E2、杂交瘤细胞株及其制备方法和应用,该杂交瘤细胞保存于中国典型培养物保藏中心,保藏号为：CCTCC NO：C2019181,该杂交瘤细胞能够分泌效价高的单克隆抗体,获得抗体能够与HPV16阳性的细胞株CaSki和HPV16阳性宫颈癌鳞癌石蜡组织切片反应,具有很好的特异性；可以作为ELISA试剂盒、免疫化学试剂盒、免疫荧光试剂盒、化学发光免疫分析检测试剂盒等的检测试剂。(The invention discloses an anti-HPV 16E7 protein monoclonal antibody 69E2, a hybridoma cell strain, a preparation method and an application thereof, wherein the hybridoma cell is preserved in China center for type culture collection with the preservation number of CCTCC NO: C2019181, can secrete monoclonal antibodies with high titer, can react with HPV16 positive cell strains CaSki and HPV16 positive cervical cancer squamous cell carcinoma paraffin tissue slices to obtain antibodies, has good specificity, and can be used as detection reagents of E L ISA kits, immunochemical kits, immunofluorescence kits, chemiluminescence immunoassay detection kits and the like.)

1. anti-HPV 16E7 protein monoclonal antibody 69E2, characterized in that: the monoclonal antibody 69E2 comprises a heavy chain and a light chain, wherein the amino acid sequences of the variable region CDR1, CDR2 and CDR3 of the heavy chain are respectively shown as the 50 th-54 th site, 69 th-85 th site and 118 th-127 th site of SEQ ID NO. 6; the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain are shown in positions 46-55, positions 71-77 and positions 110-118 of SEQ ID NO. 8.

2. The anti-HPV 16E7 protein monoclonal antibody 69E2 of claim 1, wherein: the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6; the amino acid sequence of the light chain is shown as SEQ ID NO. 8.

3. The anti-HPV 16E7 protein monoclonal antibody 69E2 of claim 1, wherein: the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5; the nucleotide sequence of the light chain is shown as SEQ ID NO. 7.

4. The anti-HPV 16E7 protein monoclonal antibody 69E2 according to any one of claims 1-3, wherein: the monoclonal antibody is secreted by a hybridoma cell strain HPV16E7-69E2, and the preservation number is CCTCC NO: C2019181.

5. hybridoma cells secreting monoclonal antibody 69E2 against HPV16E7 protein, characterized in that: the hybridoma cells are preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019181.

6. the method for preparing the monoclonal antibody 69E2 against HPV16E7 protein according to any one of claims 1 to 4, which is characterized by comprising the steps of immunizing a mouse by using HPV16E7 protein as an antigen, fusing SP2/0 with splenocytes or lymph node cells of the immunized mouse BA L B/c by using a hybridoma technology, screening a hybridoma cell strain with good stability and high cost resistance against HPV16E7 protein to prepare monoclonal antibody ascites, and purifying to obtain the monoclonal antibody 69E2 against HPV16E7 protein.

7. The use of the anti-HPV 16E7 protein monoclonal antibody 69E2 of any one of claims 2-5 in the preparation of a kit for detecting HPV16E7 protein.

8. Use according to claim 7, characterized in that: the monoclonal antibody 69E2 in the kit is used as a detection antibody.

9. A kit comprising the anti-HPV 16E7 protein monoclonal antibody 69E2 according to any one of claims 2 to 5.

10. The kit according to claim 8, wherein the kit is an E L ISA kit, an immunochemical kit, an immunofluorescence kit, a chemiluminescent detection kit or a Western Blot detection kit.

Technical Field

The invention relates to the field of immunology, in particular to a hybridoma cell secreting monoclonal antibody 69E2 against HPV16E7 protein, and also relates to a monoclonal antibody secreted by the cell, and a preparation method and application thereof.

Background

The cervical cancer is the fourth cancer among the cancers of women worldwide, the annual increasing 530,000 cases and dying 275,000 cases, about 85% of the cases are from developing countries, in China, the incidence rate of cervical cancer is the first of female genital tract malignant tumors, the continuous infection of cervical High-risk human papillomavirus (HR-HPV) is the direct cause of Cervical Intraepithelial Neoplasia (CIN) and cervical cancer, wherein HPV16 type is the highest and accounts for 53%. the HPV 7 protein is the key molecule of HPV carcinogenesis, the E7 oncogenic protein is selectively expressed in tumor cells and is not expressed in normal cells, as the cervical intraepithelial neoplasia CIN is subjected to High grade from low grade (from the incidence rate of the cervical intraepithelial neoplasia to 1 CIN2, CIN3 to erosive cervical cancer), the expression of the E7 protein is obviously advanced to the outer layer and is higher and higher in expression amount, and the final HPV 7 protein is extracted from the cervical cancer cells, the cervical cancer patients with cervical cancer-HR 2, the cervical cancer is subjected to High grade (from low grade from the cervical cancer incidence rate of cervical cancer, the cervical cancer is subjected to HPV 2, the cervical cancer is subjected to the cervical cancer, the cervical cancer detection of cervical cancer, the cervical cancer is subjected to the cervical cancer, the cervical cancer detection of the cervical cancer, the cervical cancer is subjected to the cervical cancer detection of the cervical cancer, the cervical cancer detection is the cervical cancer detection of the cervical cancer, the cervical cancer detection of the cervical cancer, the cervical cancer detection is the cervical cancer, the cervical cancer detection of the cervical cancer detection is not the cervical cancer, the cervical cancer detection of the cervical cancer, the cervical cancer detection is the cervical cancer, the cervical cancer detection is the cervical cancer detection is the detection is.

Disclosure of Invention

In view of the above, the present invention aims to provide an anti-HPV 16E7 protein mab 69E 2; the second purpose of the invention is to provide hybridoma cells secreting monoclonal antibody 69E2 against HPV16E7 protein; the invention also aims to provide a preparation method of the anti-HPV 16E7 protein monoclonal antibody 69E 2; the fourth purpose of the invention is to provide the application of the monoclonal antibody 69E2 for resisting HPV16E7 protein in preparing a kit for detecting HPV16E7 protein; the fifth purpose of the invention is to provide a kit containing the anti-HPV 16E7 protein monoclonal antibody 69E 2.

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

1. the monoclonal antibody 69E2 for resisting HPV16E7 protein, wherein the monoclonal antibody 69E2 comprises a heavy chain and a light chain, and the amino acid sequences of the variable region CDR1, CDR2 and CDR3 of the heavy chain are respectively shown as the 50 th-54 th site, 69 th-85 th site and 118 th-127 th site of SEQ ID NO. 6; the amino acid sequences of the variable regions CDR1, CDR2 and CDR3 of the light chain are shown in positions 46-55, positions 71-77 and positions 110-118 of SEQ ID NO. 8.

Preferably, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 6; the amino acid sequence of the light chain is shown as SEQ ID NO. 8.

Preferably, the nucleotide sequence of the heavy chain is shown as SEQ ID NO. 5; the nucleotide sequence of the light chain is shown as SEQ ID NO. 7.

Preferably, the monoclonal antibody is secreted by a hybridoma cell strain HPV16E7-69E2, and the preservation number is CCTCC NO: C2019181.

2. the hybridoma cells secreting monoclonal antibody 69E2 resisting HPV16E7 protein are preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2019181.

3. the preparation method of the anti-HPV 16E7 protein monoclonal antibody 69E2 comprises the following steps of immunizing a mouse by taking HPV16E7 protein as an antigen, fusing SP2/0 with spleen cells or lymph node cells of the immunized mouse BA L B/c by using a hybridoma technology, screening a hybridoma cell strain with good stability and high titer for resisting HPV16E7 protein, preparing monoclonal antibody ascites, and purifying to obtain the anti-HPV 16E7 protein monoclonal antibody 69E 2.

4. The anti-HPV 16E7 protein monoclonal antibody 69E2 is applied to the preparation of a kit for detecting HPV 16.

Preferably, monoclonal antibody 69E2 in the kit is used as a detection antibody.

5. A kit containing the anti-HPV 16E7 protein monoclonal antibody 69E 2.

Preferably, the kit is an E L ISA kit, an immunochemical kit, an immunofluorescence kit, a chemiluminescence detection kit and a Western Blot detection reagent.

The invention has the beneficial effects that: the invention discloses a hybridoma cell secreting monoclonal antibody 69E2 resisting HPV16E7 protein, which can secrete monoclonal antibody 69E2 resisting HPV16E7 protein, can be used for quantitative detection of HPV16E7 protein, has the advantages of good specificity and high sensitivity, and has important significance for early diagnosis of cervical cancer.

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 shows the result of reaction of purified HPV16E7 protein with histidine-tagged antibody by Western Blot detection.

FIG. 2 is a 10% SDS-PAGE electrophoresis of purified mAb 69E2 (M: Marker; lanes 1 and 2, two bands at molecular weights 50kDa and 25kDa, respectively).

FIG. 3 is the peak titer chart for mAb 69E 2.

FIG. 4 shows 4 pairs of mAbs that can be paired.

FIG. 5 is a graph showing affinity assay of mAb 69E 2.

FIG. 6 is a graph showing the immunocytochemistry results of mAb 69E2 with CaSki cells and He L a cells (10 × 20).

FIG. 7 is a diagram of the immunohistochemical results of the monoclonal antibody 69E2 and HPV 16-positive cervical cancer squamous cell carcinoma and HPV 18-positive cervical cancer adenocarcinoma.

FIG. 8 is a graph showing the results of indirect immunofluorescence reactions of mAb 69E2 with CaSki cells and He L a cells (10 × 40).

FIG. 9 is a graph showing the results of Western Blot reactions between mAb 69E2 and total proteins from CaSki cells and He L a cells.

FIG. 10 is a linear relationship of the antigen HPV16E7 protein diluted by the equal ratio equal difference dilution method at ng grade (A: when antigen HPV16E7 proteinDetermination coefficient R of straight line fitting of prepared reference curve when diluting at ng grade by equal ratio equal difference method (0, 25, 50, 100, 200, 400, 600, 800, 1000 ng/hole)²＝0.9723，R²The judgment coefficient is also called a decision coefficient and is also called goodness-of-fit, and is established on the basis of regression analysis and used for researching the interpretation degree of one random variable to another random variable, the value is 0-1, and the closer to 1, the higher the interpretation degree of the independent variable to the dependent variable is; b: the coefficient of variation CV is the standard deviation/average of the original data, two points with low concentration content with antigen of 0 and coefficient of variation CV lower than 10% are used as the abscissa, the corresponding OD value is used as the ordinate, the equation for obtaining the calculation detection limit is Y0.01122X +0.6896 and the judgment coefficient R²The value is 0.9889; c: S/N is equal to the OD value of the Sample to be detected/the Negative control OD value, S is Sample of the Sample to be detected, N is Negative control Negative, and the OD of the lower limit of the quantification is accurately determined by the high standard of S/N equal to 3_450nm3 × 0.6896 ═ 2.0688, lower limit of quantitation (2.0688-0.6896)/0.01122 ═ 122.9234 ng.

FIG. 11 is a graph showing a reference curve for detecting a cervical cancer specimen and the HPV16E7 protein content in the specimen (A, B, C is a graph showing straight line fitting, log-log fitting and signal-to-noise ratio of the reference curve for detecting a cervical cancer specimen, D is a reference curve and equation for calculating a detection limit, and E is an HPV16E7 content in3 groups of tissues including 4 cervical normal tissues, 12 cervical cancer tissues positive to HPV16 and 4 cervical cancer tissues positive to HPV 52).

FIG. 12 shows the epitope identification result of mAb 69E2 (A: result of indirect E L ISA method with overlapping peptides; B: result of indirect competition E L ISA method; C: location of the specific epitope of mAb 69E2 on the HPV16E7 protein three-dimensional crystal structure map).

FIG. 13 is a graph showing the conservation analysis alignment of the specific epitope of mAb 69E 2.

Deposit description

Hybridoma cell strains secreting antibodies 79A11, 69A6 and 69E2 are sent to China center for type culture Collection, the collection addresses are Wuhan university in Wuhan, China, and the hybridoma cell strains are named as HPV16E7-79A11, HPV16E7-69A6 and HPV16E7-69E2 respectively; the preservation date of HPV16E7-79A11 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201718, namely a monoclonal hybridoma cell strain HPV16E7-79A 11; the preservation date of HPV16E7-69A6 is 2017, 4 and 10 months, and the preservation number is CCTCC NO: c201720, namely a monoclonal hybridoma cell strain HPV16E7-69A 6; the preservation date of HPV16E7-69E2 is 8-21.2019, and the preservation number is CCTCC NO: c2019181, and is classified and named as a mouse hybridoma cell strain HPV16E7-69E 2.

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.

The invention clones and expresses HPV16E7 gene by gene engineering technology, takes purified HPV16E7 protein as antigen immune BA L B/c mouse, fuses spleen cells and lymph node cells of SP2/0 and immune BA L B/c mouse by hybridoma technology, screens and obtains anti-HPV 16E7 protein monoclonal hybridoma cell strain with good stability and high titer, prepares monoclonal antibody, purifies and obtains 3 monoclonal antibody with higher purity and higher titer by salting out, affinity chromatography and gel chromatography and other comprehensive methods, and is named as 79A11, 69A6 and 69E 2.