Method for constructing mammary gland specifically expressed human bactericidal/permeability-increasing protein (hBPI) vector

A protein carrier and construction method technology, applied in the field of bioengineering, can solve problems such as low success rate, low expression level, and long test cycle

Inactive Publication Date: 2012-09-19
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, the outstanding problems faced by the development of animal mammary gland bioreactors are low success rate, low expression level, long test cycle and high development cost

Method used

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  • Method for constructing mammary gland specifically expressed human bactericidal/permeability-increasing protein (hBPI) vector
  • Method for constructing mammary gland specifically expressed human bactericidal/permeability-increasing protein (hBPI) vector
  • Method for constructing mammary gland specifically expressed human bactericidal/permeability-increasing protein (hBPI) vector

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Experimental program
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Embodiment Construction

[0028] 1. Mammary gland-specific expression of human bactericidal permeability-enhancing protein (hBPI) vector construction

[0029] (1) According to the human BPI gene sequence (1464bp) (Genebank: NM_001725.2), we designed the sequence as follows: XhoI restriction site + Kozak sequence + bovine β-casein signal peptide + CDS sequence of human BPI + XhoI restriction The site was synthesized by Genscript and cloned into the EcoR V restriction site of the pUC57 vector, named pUC57-hBPI.

[0030] (2) Kit to extract pUC57-hBPI, pBC1-loxp-neo-loxp-pBD plasmids, add plasmids, restriction enzymes Xho I, Buffer, H 2 O constituted a 50 μl reaction system, and digested at 37°C for 4 hours. Gel recovery of the remaining large fragments after digestion of PBC1-PBD, and small fragments after digestion of pUC57-hBPI. The pBC1-loxp-neo-loxp-pBD digestion reaction system is shown in Table 1:

[0031] Table 1

[0032] components

volume

PBC1-PBD

24μl

10×Buffer ...

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Abstract

The invention discloses a method for constructing a mammary gland specifically expressed human bactericidal / permeability-increasing protein (hBPI) vector, belonging to the technical field of bioengineering. The method is characterized by synthesizing a CDS sequence comprising a target gene hBPI in vitro, directionally cloning the sequence synthesized in vitro to a pBC1-loxp-neo-loxp-pBD vector subjected to XhoI enzyme cutting and obtaining the correctly constructed mammary gland specifically expressed hBPI vector pBC1-loxp-neo-loxp-hBPI through bacteria liquid polymerase chain reaction (PCR) and sequencing verification, wherein an XhoI enzyme cutting site, a Kozak sequence and a bovine beta-casein signal peptide are added to the upstream of the CDS sequence; and the XhoI enzyme cutting site is added to the downstream of the CDS sequence. Functional verification at a cell C-127 level shows that the vector gene is reasonably and effectively constructed, which will lay a solid foundation for successfully preparing a mammary gland bioreactor to produce the hBPI protein.

Description

[technical field] [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for constructing a mammary gland-specific expression human bactericidal permeability-enhancing protein carrier, and the method is used for preparing a transgenic animal mammary gland bioreactor. [Background technique] [0002] Human bactericidal permeability / permeability enhancer protein (hBPI) was first isolated from the azurophilic blue granules of human neutrophils (PMN) by Weiss et al. in 1978. The full-length coding region of hBPI gene contains 1464 base pairs After translation, it consists of 456 amino acid residues and is a protein with a molecular weight of about 55-60KD. Its structure is similar to that of endotoxin-binding protein. It can bind to free LPS or Gram-negative bacterial cell outer membrane LPS, and bind to all The formed BPI-LPS complex can prevent the host cell immune response triggered by LPS, neutralize endotoxin and kill bac...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66
Inventor 张运海章孝荣桂涛李运生刘亚陈建文章美玲张宇刘星方富贵丁建平
Owner ANHUI AGRICULTURAL UNIVERSITY
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