Antihuman Reg4 monoclone antibody, preparation, application and hybrid tumor cell strain

A monoclonal antibody, fusion protein technology, applied in the direction of anti-animal/human immunoglobulin, biochemical equipment and methods, instruments, etc., can solve problems such as little understanding of Reg4 function

Inactive Publication Date: 2008-09-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But as a newly discovered gene, little is known about the func...

Method used

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  • Antihuman Reg4 monoclone antibody, preparation, application and hybrid tumor cell strain
  • Antihuman Reg4 monoclone antibody, preparation, application and hybrid tumor cell strain
  • Antihuman Reg4 monoclone antibody, preparation, application and hybrid tumor cell strain

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Experimental program
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Embodiment 1

[0029] Embodiment 1: Reg4 prokaryotic expression vector

[0030]The pReceiver-B01 / Reg4 plasmid containing the nucleotide sequence shown in SEQ ID No.3 was constructed by Guangzhou Funeng Gene Company.

Embodiment 2

[0031] Embodiment 2: Expression and purification of Reg4-his fusion protein

[0032] (1) Recombinant protein induction:

[0033] Pick a single clone colony containing the pReceiver-B01-reg4 plasmid, inoculate it in 100 ml of 2YT medium containing chloramphenicol and ampicillin, shake at 220 rpm at 37°C overnight. The next day, it was diluted 1:100 into 1000ml 2YT medium, cultured at 37°C until OD600 was between 0.5 and 1.0, and IPTG was added to make the final concentration 1 mM. Incubation was continued at 37°C for 5 hours. Centrifuge at 10,000 rpm for 20 minutes at 4° C., collect the precipitate and wash it with PBS (Phosphate Buffer Solution) phosphate buffer solution, and weigh the wet bacteria. The IPTG used in the present invention comes from the German Merck (Calbiochem) manufacturer. 2YT medium formula: Tryptone 16g, Yeast extract 10g, NaCl 5g, water 1000ml, pH7.0;

[0034] (2) Denaturation and extraction of inclusion bodies with guanidine hydrochloride:

[0035] 1...

Embodiment 3

[0053] Embodiment 3: the preparation of antiserum

[0054] Use 50 mg of purified Reg4-his protein and an equal volume of complete Freund's adjuvant to fully mix and emulsify, and the formation of water-in-oil is a sign of successful mixing. Female BALB / c mice aged 6-8 weeks were injected subcutaneously at multiple points on the back for initial immunization. Two weeks later, BALB / c mice were boosted with an equal volume of incomplete Freund's adjuvant and 50 mg of Reg4-his protein emulsified, and boosted every two weeks thereafter. After boosting immunization for 3 times, blood was collected from the eyes of the mice and the serum was separated, and the titer was determined by indirect ELISA method. When it reached 10 -4 Then ready to blend. A booster immunization was given three days before fusion.

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Abstract

The present invention provides an anti-human regenrating gene 4 monoclonal antibody, a preparation method and applications thereof as well as a hybridoma cell line producing the monoclonal antibody. The monoclonal antibody has specificity in respect to the 333th to 743th nucleotide (that is, the nucleotide sequence shown as SEQ ID No.3) encoded polypeptides of the nucleotide sequence shown as SEQ ID No.1. The present invention is mainly characterized in providing the anti-human regenrating gene 4 monoclonal antibody, the preparation method and applications thereof as well as the hybridoma cell line producing the monoclonal antibody, laying a foundation for further crystalizing the expression of Reg 4 in colorectal tissues, and the anti-human regenrating gene 4 monoclonal antibody can be used for the preparation of the early detection kit of colorectal cancer.

Description

(1) Technical field [0001] The invention relates to a monoclonal antibody against human regeneration gene 4 (Regenrating gene 4, Reg4 for short), its preparation and application, and a hybridoma cell line producing the monoclonal antibody. (2) Background technology [0002] Colorectal cancer is one of the malignant tumors that seriously endanger human health. Early diagnosis and early treatment have been recognized as the key to improving the curative effect and prognosis of colorectal cancer, and its significance is far greater than any one treatment plan. The vast majority of colorectal adenocarcinomas are cancerous adenomas. If adenomas can be detected and removed in time, it is likely to prevent the rising incidence of colorectal cancer before it happens, or fundamentally improve the cure rate. However, currently commonly used colorectal tumor markers, such as CEA, TP53, K-RAS, CD95, etc., all have the problem of incompatibility between specificity and sensitivity, espe...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/18G01N33/577
Inventor 来茂德李风英沈建根
Owner ZHEJIANG UNIV
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