阅读说明：本技术 一种抗错配修复蛋白mlh1单克隆抗体及其免疫检测应用 (Anti-mismatch repair protein MLH1 monoclonal antibody and immunodetection application thereof ) 是由 张乾坤 叶露 任琪 张冉冉 王成林 钮倩 付伟 何为无 于 2019-08-09 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种杂交瘤细胞株OTI4H4(保藏编号为CGMCC No.18199)分泌的抗错配修复蛋白MLH1的单克隆抗体。本发明还涉及OTI4H4分泌的单克隆抗体在制备用于检测错配修复蛋白MLH1的免疫检测工具中的应用,包含免疫组织化学检测试剂盒与标记肿瘤试剂盒中的应用。本发明所述单克隆抗体可与错配修复蛋白MLH1特异性结合,显著提高了错配修复蛋白MLH1免疫检测的特异性和可靠性。(The invention relates to the technical field of biology, and discloses a monoclonal antibody of an anti-mismatch repair protein MLH1 secreted by a hybridoma cell strain OTI4H4 (with the preservation number of CGMCC No. 18199). The invention also relates to application of the monoclonal antibody secreted by the OTI4H4 in preparing an immunodetection tool for detecting the mismatch repair protein MLH1, and application of the monoclonal antibody in an immunohistochemical detection kit and a tumor labeling kit. The monoclonal antibody can be specifically combined with the mismatch repair protein MLH1, and the specificity and reliability of immunodetection of the mismatch repair protein MLH1 are obviously improved.)

1. A monoclonal antibody characterized by: the antibody specifically binds to mismatch repair protein MLH1, and is secreted by hybridoma cell strain OTI4H4 with the preservation number of CGMCC No. 18199.

2. A hybridoma cell strain OTI4H4, which is characterized in that: secretes the monoclonal antibody which is combined with the specificity of the mismatch repair protein MLH1, and the preservation number of the hybridoma cell strain is CGMCC No. 18199.

3. Use of a monoclonal antibody according to claim 1 for the preparation of an immunoassay tool for the detection of the mismatch repair protein MLH 1.

4. The use according to claim 3, wherein the immunoassay means is a kit, chip or strip.

5. An immunohistochemical detection kit comprising the monoclonal antibody of claim 1.

6. Use of the monoclonal antibody of claim 1 for the preparation of a kit for labeling tumor cells and tumor tissue.

7. The use according to claim 6, wherein the tumor cells include a colon cancer cell line (LoVo), an MLH 1-deficient colon cancer cell line (HCT116), a renal cancer cell line (TK-10), a breast cancer cell line (MCF-7), a prostate cancer cell line (Lncap), and a cervical cancer cell line (Hela); tumor tissues include colon cancer, MLH1 deficient colon cancer, breast cancer, prostate cancer, thyroid cancer, lymphoma, and pancreatic cancer.

Technical Field

The invention relates to the technical field of biology, in particular to a method for specifically combining a monoclonal antibody secreted by a hybridoma cell strain OTI4H4 with a mismatch repair protein MLH1 for immunodetection and application thereof.

Background

The mismatch repair system (MMR) is a fundamental guarantee of accurate replication and stability of intracellular genomes. MMR proteins play an important role in repair mechanisms, recognize mismatched bases occurring during DNA replication, and undergo hydrolysis and repair, thereby maintaining the stability of genetic material. However, MMR gene mutation or promoter methylation can cause the occurrence of tumors due to the loss of mismatch repair function of body cells and the instability of genome.

MutL homolog 1(MutL homolog1, MLHl) is one of the important members of the mismatch repair gene family, and plays an important role in maintaining the integrity of genetic information and avoiding the generation of genetic mutations. The MLHl gene is positioned at 3p21.3 and is a high-density distribution region of DNA mismatch repair, and methylation of a promoter region causes the expression of the MLHl gene to be silenced, thereby causing the inactivation of the mismatch repair function. Research shows that MLHl is expressed in various tumor tissues, and functional defects of MLHl are considered to be important mechanisms of tumor pathogenesis and become hot spots of research in recent years. When the MLHl gene promoter is hypermethylated, the expression level is reduced, the base mismatch repair function of DNA is reduced, errors occur in the DNA replication process, and finally tumors are induced. The MLHl protein deletion causes that tumor cells are difficult to respond to the influence of DNA damage on the tumor cells, and the malignancy degree of the tumor caused by MMR system function deletion is lower.

At present, the expression condition of mismatch repair protein MLH1 in tumor tissues is clinically detected mainly through Immunohistochemistry (IHC for short), and the quality of the performance directly determines the sensitivity and specificity of the whole detection. Therefore, the development of a monoclonal antibody aiming at the mismatch repair protein MLH1 with higher binding specificity has important significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a monoclonal antibody specifically binding to mismatch repair protein MLH1, and its application in the preparation of an immunoassay tool for detecting mismatch repair protein MLH 1.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a hybridoma cell strain OTI4H4 capable of secreting monoclonal antibodies against GPA33 protein, which is preserved in China general microbiological culture Collection center (CGMCC for short), the preservation date is 7 and 11 days in 2019, and the preservation number is CGMCC No. 18199.

The invention also provides a monoclonal antibody specifically combined with the mismatch repair protein MLH1, which is secreted by the hybridoma cell strain OTI4H 4.

The preparation method of the monoclonal antibody comprises the following steps:

(1) construction of recombinant expression vectors: the nucleotide sequence of the gene synthesis MLH1 is shown as SEQ ID No.1, the ORF length is 660bp, the corresponding mismatch repair protein MLH1 comprises the 339-558 amino acid sequence, and the amino acid length is 220aa as shown as SEQ ID No. 2. The ORF of the synthetic mismatch repair protein MLH1 was inserted into the expression vector pET23a-His (purchased from Origene Co.), and a recombinant expression plasmid pET23a-His-rMLH1 of the mismatch repair protein MLH1 was constructed.

(2) Expression and purification of the recombinant mismatch repair protein MLH 1: transforming the constructed mismatch repair protein MLH1 recombinant expression plasmid into E.coli cells, cracking and centrifuging to obtain soluble protein, and purifying by a nickel column affinity chromatography column to obtain the purified recombinant mismatch repair protein MLH 1.

(3) Screening of monoclonal antibody hybridoma and preparation of monoclonal antibody secreted by the monoclonal antibody: immunizing a BALB/c mouse by adopting the recombinant mismatch repair protein MLH1, fusing spleen cells of the mouse with SP2/0 cells, obtaining a monoclonal by a limiting dilution method, screening positive hybridoma cells by an ELISA method, obtaining a specific antibody hybridoma cell strain capable of secreting the anti-mismatch repair protein MLH1, and performing subtype identification; preparing the antibody through serum-free culture medium, and purifying through an affinity chromatography column to obtain the anti-mismatch repair protein MLH1 monoclonal antibody. The sensitivity and specificity of the monoclonal antibody is verified by immunodetection methods such as Western Blot (WB), immunohistochemical experiments (IHC).

After the preparation by the method, the hybridoma capable of secreting the anti-mismatch repair protein MLH1 monoclonal antibody is screened out and named as OTI4H4, the subtype is identified as IgG2a, and the hybridoma is preserved in China general microbiological culture Collection center (CGMCC for short) in 2019, 7 and 11 months, and the preservation number is CGMCC No. 18199.

The invention also provides application of the monoclonal antibody secreted by the hybridoma cell strain OTI4H4 in preparation of an immunodetection tool for detecting the mismatch repair protein MLH 1.

Preferably, the immunoassay tool is a kit, a chip or a test paper.

The invention also provides an immunohistochemical detection kit, which comprises an anti-mismatch repair protein MLH1 monoclonal antibody secreted by the hybridoma cell strain OTI4H4 and can detect the expression condition of the mismatch repair protein MLH1 in tissue cells.

In addition, the invention also provides application of the anti-mismatch repair protein MLH1 monoclonal antibody secreted and generated by the hybridoma cell strain OTI4H4 in preparation of a labeled tumor cell kit.

Preferably, the tumor cells include a colon cancer cell line (LoVo), an MLH 1-deficient colon cancer cell line (HCT116), a renal cancer cell line (TK-10), a breast cancer cell line (MCF-7), a prostate cancer cell line (Lncap), and a cervical cancer cell line (Hela); tumor tissues include colon cancer, MLH1 deficient colon cancer, breast cancer, prostate cancer, thyroid cancer, lymphoma, and pancreatic cancer.

The monoclonal antibody which can be stably secreted by a hybridoma cell strain OTI4H4 and is specifically combined with the mismatch repair protein MLH1, so that the specificity, accuracy and reliability of the mismatch repair protein MLH1 immunodetection are obviously improved, and the monoclonal antibody is widely suitable for marking the mismatch repair protein MLH1 in various tumors.

Biological preservation information description

The hybridoma cell strain OTI4H4 for preserving and secreting the anti-mismatch repair protein MLH1 monoclonal antibody is classified and named as: mouse anti-human mismatch repair protein MLH1 monoclonal hybridoma cell strain

The preservation unit is called as follows: china general microbiological culture Collection center

The preservation unit is abbreviated as: CGMCC (China general microbiological culture Collection center)

The address of the depository: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang

The preservation date is as follows: 7 month and 11 days 2019

The preservation number is: CGMCC No.18199

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the ORF cloning site design of MLH1 in example 1.

Fig. 2 is a graph showing the results of WB detection of the recombinant mismatch repair protein MLH1 in example 2, using anti-His to detect the expression of the recombinant mismatch repair protein MLH1 in e. Wherein, the Lane L is the detection result of E.coli cell lysate transfected with empty vector as antigen; lane R shows the detection of E.coli cell lysate antigen transfected with pET23a-His-rMLH1 plasmid.

FIG. 3 is a diagram showing the results of detection of the recombinant mismatch repair protein MLH1 by SDS-PAGE in example 2, wherein the recombinant mismatch repair protein MLH1 was purified by a nickel affinity chromatography column, and the purified protein was stained by SDS-PAGE and Coomassie blue staining.

FIG. 4 is a graph showing the results of detecting the expression of the mismatch repair protein MLH1 in various cancer cell line protein lysates by the monoclonal antibody WB secreted from OTI4H4 in example 4. Among them, lanes 1 to 6 are the results of detection of lysates from human colon cancer cell line (LoVo), MLH1 deficient colon cancer cell line (HCT116), renal cancer cell line (TK-10), breast cancer cell line (MCF-7), prostate cancer cell line (Lncap) and cervical cancer cell line (Hela), respectively, as antigens.

FIG. 5 is a graph showing the results of measuring the expression distribution of mismatch repair protein MLH1 in intestinal cancer tissues by the monoclonal antibody IHC secreted by OTI4H4 in example 5, and arrows indicate intestinal cancer cells positively expressed by the mismatch repair protein MLH1 (primary antibody against the mismatch repair protein MLH1 monoclonal antibody secreted by OTI4H4, 1. mu.g/ml).

FIG. 6 is a graph showing the results of measuring the expression distribution of the mismatch repair protein MLH1 in MLH 1-deleted intestinal cancer tissues by the monoclonal antibody IHC secreted by OTI4H4 in example 5, and arrows indicate MLH-deleted intestinal cancer cells (primary antibody against the mismatch repair protein MLH1 monoclonal antibody secreted by OTI4H4, 1. mu.g/ml).

FIG. 7 is a graph showing the comparison of the sensitivity of detecting the expression of the mismatch repair protein MLH1 in tissues of breast cancer, prostate cancer, thyroid cancer, lymphoma and pancreatic cancer by using the OTI4H4 secreted monoclonal antibody and ES05 antibody IHC in example 6 (primary antibody against the mismatch repair protein MLH1 secreted by OTI4H4, 1. mu.g/ml; ES05 antibody, 226. mu.g/ml).

Detailed Description

The invention discloses a method for using an anti-mismatch repair protein MLH1 monoclonal antibody secreted by a hybridoma cell strain OTI4H4 for immunodetection and application. The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. Based on the embodiments of the present invention, those skilled in the art can appropriately modify the process parameters to implement the embodiments based on the contents of the present invention. It is particularly pointed out that all other embodiments obtained by the person skilled in the art without making any inventive step belong to the scope of protection of the present invention.