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HPV58E6 specificity rabbit monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and specific technology, which is applied in the fields of medicine and bioengineering to achieve the effects of high sensitivity, high detection specificity and shortened time

Active Publication Date: 2015-05-13
ATTACHED OBSTETRICS & GYNECOLOGY OSPITAL MEDICALCOLLEGE ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently there is only HPV16-E6 antibody on the market, but no HPV58-E6 antibody

Method used

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  • HPV58E6 specificity rabbit monoclonal antibody as well as preparation method and application thereof
  • HPV58E6 specificity rabbit monoclonal antibody as well as preparation method and application thereof
  • HPV58E6 specificity rabbit monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] This embodiment prepares HPV58E6 specific rabbit monoclonal antibody, comprises the following steps:

[0026] Step 1, peptide synthesis and cross-linking

[0027] Search out the sequences of HPV58E6, HPV16E6, HPV31E6, HPV33E6, HPV52E6, HPV67E6 from http: / / www.ncbi.nlm.nih.gov / pubmed / , and compare HPV58E6 with HPV16E6, HPV31E6, HPV33E6, HPV52E6, HPV67E6 sequences, find out For the specific sequence of HPV58E6, two polypeptides are designed for the specific sequence, which are specific HPV58E6 polypeptide antigens ZJM-1a and ZJM-1b; the sequences of the two specific HPV58E6 polypeptide antigens are VCWRPRRRQTQV (SEQ ID NO.5) and ETSVHEIELKCV (SEQ ID NO. 6).

[0028] Step 2. Immunization of animals and serum titer determination

[0029] Immunization scheme: standard immunization scheme is used for immunization, each rabbit is given 5-6 injections for immunization, and serum is collected 2-3 times for testing. For each injection of immunization, thaw the aliquoted antige...

Embodiment 2

[0057] In this example, the HPV58E6-specific rabbit monoclonal antibody prepared in Example 1 was applied to detect HPV58E6 protein. The sequences of HPV58E6 and HPV16E6 were respectively constructed into the pEGFP-C1 plasmid vector, the pEGFP-C1 empty plasmid and the plasmid containing HPV58E6 and HPV16E6 were transfected into 293T cells, the cell protein was extracted after 48 hours, and the obtained protein was used as the sample to be tested. The groups were divided into blank control group, pEGFP-C1 empty plasmid group, pEGFP-C1-HPV58E6 plasmid transfection group, and pEGFP-C1-HPV16E6 plasmid transfection group. Detection of HPV58E6 protein expression by western blot, the results are as follows Figure 5 , it can be seen from the figure that obvious bands can be seen in the HPV58E6 group between 40 and 55KD, but no bands are seen in the other groups. It can be seen that the HPV58E6-specific rabbit monoclonal antibody prepared in Example 1 can be applied to detect HPV58E6...

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Abstract

The invention relates to a preparation method of an HPV58E6 specificity rabbit monoclonal antibody. The preparation method comprises the following steps: designing an HPV58E6 specificity polypeptide segment; synthesizing the polypeptide segment and crosslinking with two vectors; immunizing a New Zealand rabbit with the synthesized polypeptide antigen, measuring serum titer and detecting the antibody specificity through Western Blot; then carrying out cell fusion culture on splenocyte and fusion partner cells; taking supernatant and detecting antibody titer and specificity; screening out fusion cells with the best specificity and titer; carrying out fusion cell lysis to extract RNA, and carrying out RT-PCR to obtain cDNAs of heavy and light chains of the antibody so as to establish a pTT5 plasmid vector; and co-transfecting the plasmid containing the heavy and light chains to an HEK-293-6E cell to be expressed to finally obtain a recombinant antibody, namely HPV58-E6 monoclonal antibody, wherein the antibody can be applied to Western Blot for detecting expression of HPV58 type oncogene E.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a HPV58E6 specific rabbit monoclonal antibody and its preparation method and application, belonging to the field of medicine. Background technique [0002] Cervical cancer is currently one of the leading causes of death among women in developing countries. Human papillomavirus (HPV) infection is one of the necessary factors leading to cervical cancer, which can be divided into high-risk and low-risk types according to its carcinogenicity. Human papillomavirus is a small DNA tumor virus with high species specificity, which can cause cervical lesions by infecting cervical basal epithelium. Persistent high-risk human papillomavirus (HPV) infection has been considered as the main pathogenic factor of cervical cancer and its precancerous lesions. There are 15 known high-risk types of HPV. HPV58 is a high-risk type of HPV, which is less common than HPV16 and HPV18. %. Mainland China, ...

Claims

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Application Information

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IPC IPC(8): C07K16/08G01N33/68G01N33/577
Inventor 吕卫国邹健程晓东李阳章鉴洋谢幸
Owner ATTACHED OBSTETRICS & GYNECOLOGY OSPITAL MEDICALCOLLEGE ZHEJIANG UNIV
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