Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof

An anti-human papilloma, L1 protein technology, applied to anti-human papilloma virus L1 protein antibody and its encoding gene and application field, can solve problems such as uncontrolled proliferation of epithelial cells, and achieve the effect of high affinity

Active Publication Date: 2014-04-02
HANGZHOU DALTON BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that the E6 and E7 proteins encoded by certain HPV types E6 and E7 combine with the tumor suppressor

Method used

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  • Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof
  • Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof
  • Anti-human papilloma virus L1 protein antibody, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Screening of anti-human papillomavirus L1 protein single-chain antibody

[0035] The method disclosed in the Chinese patent literature with the publication number CN1444651A is used to construct a human single-chain antibody library, and to screen the anti-human papillomavirus L1 protein single-chain antibody in the human single-chain antibody library. The specific implementation process is as follows:

[0036] 1 Amplification of human antibody heavy chain and light chain variable region DNA

[0037] Using poly A+RNA (purchased from Clontech) from human bone marrow, human fetal liver, human spleen and human peripheral blood leukocytes as templates, using oligo (dT) and random primers (random primers), using reverse transcriptase kit (purchased from From Clontech), poly A+ RNA was reverse transcribed into cDNA according to the method guide provided with the Clontech kit.

[0038] Using the above cDNA as a template, use a series of primers that recognize the hu...

Embodiment 2

[0137] Example 2 Expression of recombinant human papillomavirus HPV16 type L1 protein and preparation of rabbit antiserum

[0138] The preparation of recombinant human papillomavirus HPV16 type L1 protein refers to Peng Qinglin et al [Peng Qinglin, Zhang Ting, Fan Dongsheng, Zheng Wei, Xie Xixiu, Xu Yufei, Xu Xuemei (2009) "Expression and purification of HPV18L1 virus-like particles and preparation of guinea pig antiserum. "Basic Medicine and Clinics, 29:1039-1043]. Briefly: the HPV16 type L1 coding DNA fragment was adjusted according to the codon frequency of sf9 insect cells to optimize the codons of the HPV16 type L1 coding DNA, and the optimized coding DNA was inserted into the Xba I site of the pFastBac Dual vector to obtain the recombinant vector pFastBac- HPV16L1. Recombinant Bacmid was obtained by transforming Escherichia coli DH10Bac competent cells. Insect sf9 cells were transfected with recombinant Bacmid, cultured in a constant temperature incubator at 27°C for 7...

Embodiment 3

[0141] Embodiment 3 specific detection

[0142] 1 Single-chain antibody expression and purification

[0143] The gene encoding the single-chain antibody #H16L1-B was cloned into the expression vector pET27b(+), and pET27b-H16L1b was constructed;

[0144] Transform pET27b-H16L1b into the expressing bacteria E.coli BL21(DE3), and induce expression with IPTG (0.5mM) according to the method provided by Novagen; in the expressed target protein, the N-terminal of the scFv is the pelB sequence, which can be The expressed scFv is secreted into the periplasmic space of BL21(DE3); the C-terminus of the scFv contains an HSV tag and a 6×His tag to facilitate the purification of the target protein;

[0145] The method provided by Qiagen was conveniently separated and purified with Ni-NTA column to obtain the single-chain antibody against human papillomavirus HPV16 type L1 protein.

[0146] 2ELISA testing the specificity of single chain antibody to HPV16 type L1 protein

[0147] The HPV1...

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Abstract

The invention discloses an anti-human papilloma virus L1 protein antibody, and a coding gene and application thereof. The anti-human papilloma virus L1 protein antibody comprises a heavy chain variable region and a light chain variable region. The anti-human papilloma virus L1 protein antibody is characterized in that amino acid sequences of three hypervariable regions CDRH1, CDRH2 and CDRH3 of the heavy chain variable region are respectively disclosed as SEQ ID NO.5-7; and amino acid sequences of three hypervariable regions CDRL1, CDRL2 and CDRL3 of the light chain variable region are respectively disclosed as SEQ ID NO.8-10. The invention also discloses application of the anti-human papilloma virus L1 protein antibody in preparing reagents and medicines for detecting, preventing and treating human papilloma virus related diseases. The anti-human papilloma virus L1 protein antibody has high affinity with human papilloma virus L1 protein, and has favorable specificity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-human papillomavirus L1 protein antibody and its coding gene and application. Background technique [0002] Human papillomavirus (HPV) is a species-specific epitheliophilic virus, which is a small DNA virus with double-stranded closed circle. The HPV virus consists of a coat of protein capsid and a double-stranded circular DNA core wrapped around it. The protein capsid consists of a major capsid protein (L1) and a minor capsid protein (L2). In 1949, Strauss first observed HPV particles under the electron microscope, which were spherical, 20-hedral symmetry, and about 45-55nm in diameter. [0003] The genome of HPV consists of approximately 8000 base pairs. The HPV genome includes 8 early open reading frames (Open Reading Frame, referred to as ORF) (namely E1--E8), 2 late reading frames (ORF) and 1 non-coding long control region. In the early op...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13A61K39/42A61P31/20A61P35/00G01N33/569
Inventor 吕卫国程晓东华绍炳侯伟胡杰锋吴敏
Owner HANGZHOU DALTON BIOSCI
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