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Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies

A technology of human papillomavirus and monoclonal antibody, which is applied in the direction of antiviral immunoglobulin, microbial-based methods, and the use of vectors to introduce foreign genetic materials, etc., which can solve the problems of long time-consuming, high antibody titer, and difficulty in promotion. , to achieve the effect of improving the detection rate, strong specificity, and easy promotion

Inactive Publication Date: 2014-06-18
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In 2011, KS Anderson found that antibodies against E1, E2, E4, E6, E7 and L1 proteins of HPV16 could be detected in the serum of HPV16-related head and neck cancer patients, but antibodies against E5 and L2 proteins were not detected. The antibody titers of E1 and E7 proteins are above 1:1000 at the highest, and the others are at 1:640
However, the above methods for detecting HPV have a common limitation: it takes a long time, requires professional equipment and technical personnel, the operation procedure is complicated, the cost is high, and it is not easy to promote

Method used

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  • Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies
  • Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies
  • Monoclonal antibodies for resisting high-risk human papillomavirus proteins and application of monoclonal antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Example 1 Primer design

[0152] The software analyzes the sequences of the vector pET-28a (+), HPV16E6 gene and HPV18E7 gene and then designs primers for the full-length gene, which contains 6 histidine tags and NcoI (base sequence is ccatgg) and HindIII (base sequence is aagctt ) restriction enzyme site, 2 pairs of primers directed at the HPV16E6 gene and the HPV18E7 gene of the vector pET-28a (+), their sequences are as follows:

[0153] Primers for the E6 gene of HPV16:

[0154] Upstream primer P1 (SEQ ID NO: 1): 5'-catg- ccatggat cac cat cac cat cac atg caccaa aag aga act gca atgt-3';

[0155] Downstream primer P2 (SEQ ID NO:2): 5'-ccg- ctcgag tta cag ctg ggt ttc tct acg tgtt-3'.

[0156] Primers for the E7 gene of HPV18:

[0157] Upstream primer P1 (SEQ ID NO: 3): 5'-gc- ccatgg at cac cat cac cat cac atg catgga cct aag gca ac-3';

[0158] Downstream primer P2 (SEQ ID NO: 4): 5'-ccg- ctcgag tta ctg ctg gga tgc aca cca gg-3'.

Embodiment 2

[0159] Example 2 Construction of recombinant pET-28a(+)-HPV16E6, HPV18E7-BL21star-DE3plysS

[0160] 1. The target genes HPV16E6 and HPV18E7 were amplified by PCR using the total DNA extracted from CaSki and HeLa cells as templates. The two gene fragments were firstly subjected to gradient PCR at an annealing temperature of 55-70°C. The reaction conditions were as follows:

[0161] Pre-denaturation at 95°C for 5 minutes

[0162]

[0163] 72°C extension for 7 minutes

[0164] The PCR reaction conditions when a large amount of 2 gene fragments are amplified:

[0165] HPV16E6 gene reaction conditions:

[0166] Pre-denaturation at 95°C for 5 minutes

[0167]

[0168] 72°C extension for 7 minutes

[0169] HPV18E7 gene reaction conditions:

[0170] Pre-denaturation at 95°C for 7 minutes

[0171]

[0172] 72°C extension for 7 minutes

[0173] The 10 annealing temperatures (55.1, 56.3, 57.7, 59.4, 61.4, 63.3, 65.3, 67.6, 69.0, 69.7°C) of the 2 gene fragments of HPV16...

Embodiment 3

[0182] Example 3 Expression and Purification of Recombinant pET-28a(+)-HPV16E6 / HPV18E7-BL21star-DE3plysS

[0183] Transform the correct recombinant plasmid prepared in Example 2 to express the target protein in the competent Escherichia coli BL21star-DE3plysS for expression, because the expressed target protein has 6 histidine tags, so use nickel column affinity chromatography Purify to obtain a relatively pure target protein. The expression results are as follows:

[0184] 1. Expression results of pET-28a(+)-HPV18E7-2-BL21star-DE3plysS-1

[0185] Please refer to Figure 12 , Lane 1 is pET-28a(+)-HPV18E7-2-BL21star-DE3plysS-1 expressed in ZYM-5052 at 37°C for 18 hours, and lanes 2, 3, and 4 were expressed in 0.5mmol / L IPTG for 4 hours solution, supernatant and precipitate. It can be seen that a band with the same molecular weight as the HPV18E7 protein appears at about 13KDa, and it appears in the supernatant, not in the precipitate. The above results show that HPV18E7...

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Abstract

The invention relates to monoclonal antibodies for resisting high-risk human papillomavirus proteins HPV16E6 and HPV18E7, a hybridoma cell strain secreting the monoclonal antibodies and the application of the monoclonal antibodies. The monoclonal antibodies can be used for specifically detecting the proteins HPV16E6 and HPV18E7. The two antibodies can be prepared into immunochromatographic test strips for rapidly detecting the proteins HPV16E6 and HPV18E7 by virtue of labeled colloidal gold or color latex particles. The monoclonal antibodies for detecting the proteins HPV16E6 and HPV18E7 have the characteristics of high speed (results can be obtained within 10 minutes), simplicity, specificity, sensitivity, low cost and easiness in popularization.

Description

technical field [0001] The invention relates to a monoclonal antibody against high-risk human papillomavirus HPV16 type E6 protein and HPV18 type E7 protein and application thereof. Background technique [0002] HPV is a group of non-enveloped, circular double-stranded DNA viruses with a symmetrical shape of 20 polyhedrons. HPV belongs to the Papillomavirus A subgroup of Papovaviridae with 72 capsids, with a diameter of about 45nm to 55nm and a molecular weight of 5×10 6 dalton. The HPV genome contains 7800-7900 base pairs (bp), including 4 parts: the early transcription region, the late transcription region, the upstream regulatory region and a small highly variable non-coding region between E5 and L2, encoded as 9 open reading frames. The early transcription region, also known as the E region, consists of 4500 base pairs, which are encoded as 8 early proteins, including E1, E2, E3, E4, E5, E6, E7, and E8 (some HPVs do not contain E3, E8), It has the functions of partic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C12N15/63C07K16/08G01N33/68G01N33/569G01N33/577G01N33/558C12R1/91
Inventor 胡仁建范开蔡家利罗佳
Owner CHONGQING UNIV OF TECH
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