HPV16E7 protein nano-antibody as well as preparation method and application thereof

A HPV16E7, 1.HPV16E7 technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of poor antibody stability, high production cost, and limited papillomavirus detection.

Active Publication Date: 2017-02-15
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, traditional antibodies have poor stability and hig

Method used

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  • HPV16E7 protein nano-antibody as well as preparation method and application thereof
  • HPV16E7 protein nano-antibody as well as preparation method and application thereof
  • HPV16E7 protein nano-antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Induced expression and purification of HPV16 E7 protein

[0034] (1) Induced expression of HPV16E7 protein

[0035] First, transform the successfully constructed pSUMO-HPV16E7 vector into BL21 (DE3) to obtain pSUMO-HPV16E7 recombinant bacteria, select a single clone in kanamycin liquid LB, and culture overnight; then inoculate at a ratio of 1:100 Incubate 1.5-2h in mycin liquid LB, when the bacteria liquid OD 600 When it is 0.6-0.8, induce expression with 23°C, 16h, 0.1mmol / L IPTG.

[0036] figure 1 SDS-PAGE electrophoresis detection results showed that the induced HPV16 E7 protein expression bacteria had a band at 35KD, which was in line with the expected molecular weight of the target protein.

[0037] (2) Purification of HPV16 E7 protein

[0038] Purified HPV16E7 protein was obtained by Ni-IDA affinity chromatography.

[0039] The chromatographic column was first washed with bacterial cell buffer, and Ni-IDA was added to fill the chromatographic c...

Embodiment 2

[0041] Embodiment 2: Construction of HPV16 E7 protein Nanobody library

[0042] (1) HPV16 E7 protein immunization of Bactrian camels in Xinjiang

[0043] Mix 1 mg of HPV16 E7 protein with an equal volume of Freund's adjuvant, immunize Xinjiang Bactrian camels (subcutaneous injection at 3-5 points), and immunize 7 times (complete Freund's adjuvant for the first time, and incomplete Freund's adjuvant for the rest). Adjuvant); After the immunization, 200ml of peripheral blood from the immunized camel was taken to separate and obtain peripheral blood lymphocytes.

[0044] (2) Separation of peripheral blood lymphocytes and extraction of total RNA

[0045]Firstly, Percoll density gradient centrifugation was used to separate and purify camel peripheral blood lymphocytes. After washing the lymphocytes with normal saline for several times, add Trizol and let it stand at room temperature for 10 minutes; then add chloroform to shake vigorously and let it stand at room temperature for 1...

Embodiment 3

[0079] Example 3: Panning HPV16E7 nanobody using nickel ion metal chelation affinity chromatography medium Ni-NTA.

[0080] (1) Cleaning of Ni-NTA medium:

[0081] Take 100ul of Ni-NTA medium in a 1.5ml EP tube, add 1ml of sterilized water, and mix with a vortex shaker; centrifuge at 3000rpm for 30s, discard the supernatant; wash 5 times in total, and replace the sterilized water with 0.05% TBST for the last time.

[0082] (2) closed:

[0083] Add 1ml of blocking solution (0.5% BSA) to the washed Ni-NTA medium, turn over and shake for 1 hour; after the blocking is completed, wash with 1ml TBST for 4 times.

[0084] (3) Negative screening to remove non-specifically bound phages:

[0085] The VHH-T7 phage library of HPV16E7 was diluted to 1ml with TBST, added to the blocked Ni-NTA medium, placed on an inversion shaker, and combined at room temperature for 30 minutes; centrifuged, and the supernatant was the T7 phage library after negative screening.

[0086] (4) Screening of ...

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Abstract

The invention discloses an HPV16E7 protein nano-antibody as well as a preparation method and application thereof. According to the preparation method, camels are immunized by virtue of an HPV16E7 antigen expressed in a prokaryotic manner so as to obtain a high-quality immune nano-antibody gene pool; and an enzyme label plate is coated with HPV16E7 proteins, the immune nano-antibody gene pool is screened by virtue of a phage display technique to obtain nano-antibody genes with the HPV16E7 specificity, and then the nano-antibody genes are transferred to escherichia coli, so as to establish nano-antibody strains capable of realizing high-efficiency expression in the escherichia coli.

Description

technical field [0001] The invention belongs to the technical field of HPV detection, and in particular relates to an immunological detection method for diseases related to HPV16 infection. Background technique [0002] Human papillomavirus (HPV) is a symmetrical icosahedron, which is a small non-enveloped circular double-stranded DNA virus with a size of about 8 kb. Epidemiological surveys have confirmed that HPV infection is closely related to the occurrence of cervical cancer. The test results show that the positive rate of HPV virus genome in cervical cancer cases is greater than 99%, and 70% of the cases are caused by high-risk viruses HPV16 and HPV18. Caused by infection. HPV16 is the most common high-risk virus associated with cervical cancer. [0003] The HPV genome is divided into three regions according to different functions: the non-coding region, which is the gene transcription and replication regulatory region; the early region, which contains 6 open reading ...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/70G01N33/569
CPCG01N33/56983C12N15/70C07K16/084C07K2317/565C07K2317/569C07K2317/567G01N2333/025C12N2800/101
Inventor 李淑锋单海涛姜昆鹏马芳
Owner SOUTHEAST UNIV
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