Nano antibody capable of specifically combining HPVL6-E6 protein as well as coding sequence, preparation method and application thereof

A nano-antibody and specific binding technology, applied in the field of immunology, can solve the problems of high equipment requirements and high false positive rate of test results, and achieve high specificity and high detection sensitivity

Active Publication Date: 2019-01-15
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the clinical diagnosis related to HPV16 infection mainly relies on molecular biology methods based on PCR technology, which has the disadvantages of high equipment requirements and high false positive rate of test results.

Method used

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  • Nano antibody capable of specifically combining HPVL6-E6 protein as well as coding sequence, preparation method and application thereof
  • Nano antibody capable of specifically combining HPVL6-E6 protein as well as coding sequence, preparation method and application thereof
  • Nano antibody capable of specifically combining HPVL6-E6 protein as well as coding sequence, preparation method and application thereof

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Experimental program
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Embodiment 1

[0031] 1 Expression and purification of HPV16 E6 protein

[0032] (1) Expression of HPV16 E6 protein

[0033] Transform the constructed pSUMO-HPV16E6 vector into BL21(DE3), pick a single clone and culture overnight; then expand the culture and induce expression with IPTG.

[0034] (2) Purification of HPV16 E6 protein

[0035] Purified HPV16E6 protein was obtained by Ni-IDA affinity chromatography. figure 1 The results of SDS-PAGE electrophoresis showed that the purity of the HPV16E6 protein purified by the nickel column was as high as 90%.

[0036] Construction of 2HPV16E6 Protein Nanobody Library

[0037] (1) HPV16 E6 protein immunization of Bactrian camels in Xinjiang

[0038] Bactrian camels in Xinjiang were routinely immunized with HPV16 E6 protein, with a total of 7 immunization injections; after the immunization, 200 mL of peripheral blood was collected from the camels.

[0039] (2) Separation of peripheral blood lymphocytes and extraction of total RNA

[0040] Per...

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Abstract

The invention discloses a nano-antibody capable of specifically combining HPVL6-E6 protein as well as a coding sequence, a preparation method and application thereof, an antibody VHH chain comprises aframework region and a complementarity determining region; the framework region comprises the following four amino acid sequences: FR1. SEQ ID NO: 1, FR2, SEQ ID NO: 3, FR3, SEQ ID NO: 5, FR4 and SEQID NO: 7; the complementarity determining region comprises the following three amino acid sequences: CDR1, SEQ ID NO: CDR2, SEQ ID NO: 4, CDR3, SEQ ID NO: 6. Compared with the prior art, the advantages are as follows: the nano-antibody capable of specifically binding to the HPV16-E6 protein has high specificity and high detection sensitivity; and the antibody can be efficiently expressed in escherichia coli, so that a foundation is provided for the mass production of a subsequent immune reagent and a tumor drug composition.

Description

technical field [0001] The invention belongs to the technical field of immunology, and relates to a novel HPV16-E6 antibody or a functional fragment thereof, specifically a nanobody capable of specifically binding to the HPV16-E6 protein, its coding sequence, preparation method and application. Background technique [0002] Epidemiological surveys have confirmed that HPV infection is closely related to the occurrence of cervical cancer. HPV16 is the most common high-risk virus associated with cervical cancer. The E6 gene belongs to the early HPV gene, and the E6 protein is a small molecular basic protein with a zinc finger structure. E6 protein can combine with various proteins of host cells through the zinc finger structure to cause malignant transformation of cells and play an important role in the carcinogenic process of HPV. [0003] In 1993, Hamers Casterman of Belgium reported the existence of heavy chain antibodies lacking light chains in camel serum. Has good and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/13G01N33/569A61K39/42A61P35/00A61P31/20
CPCA61K2039/505A61P31/20A61P35/00C07K16/084C07K2317/22C07K2317/565C07K2317/567C07K2317/569C07K2317/92G01N33/56983
Inventor 李淑锋张微单海涛
Owner SOUTHEAST UNIV
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