Human membrana cofactor protein mini-gene pmcp constructing method

A technology of synergistic factors and construction methods, applied in the field of pathogenic biology, which can solve the problems of unsuccessful development of transgenic animal models, etc.

Inactive Publication Date: 2008-05-07
YANGZHOU UNIV
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Problems solved by technology

[0004] However, transgenic animal models for many human-specific p

Method used

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  • Human membrana cofactor protein mini-gene pmcp constructing method

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Embodiment Construction

[0018] Construction process of the present invention as shown in Figure 1, is divided into the following steps:

[0019] 1. Construction of receptor MCP minigene

[0020] (1) Utilize PCR technology, take human blood cell genomic DNA as the template to amplify the promoter of MCP:

[0021] The amplified upstream and downstream primer sequences are as follows:

[0022] 5′-GC ACGCGT GGTAACTGCCAGAAATTCTTTA-3' and 5'-GTACAGCAGCAACA CCATGG-3', the 5' end of the upstream primer was introduced into the Mlu I restriction site.

[0023] 50μl PCR amplification system includes: 5μl 10× buffer, 2mmol / L Mg 2+ , 200μmol / LdNTPs, 200nmol / L forward and reverse primers, 2μg human blood cell genomic DNA, 2.5U LA TagDNA polymerase. The PCR program for 30 cycles is 94°C / 50s (the first cycle is 5min), 57°C / 40s, 72°C / 2min (the last cycle is 10min). The amplified products were analyzed by electrophoresis on 1% agarose gel and recovered and purified.

[0024] (2) Using PCR technology, using pSG5-...

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Abstract

The invention relates to a construction method for human membrane cofactor protein mini gene, belonging to pathogenic biology field, which is characterized that a human blood cell genome DNA is selected as a template, and a promoter of the human membrane cofactor protein gene is cloned with a PCR method; a human membrane cofactor protein mini gene cDNA is amplified with pSG5-MCP template; then the MCP promoter is spliced with the cDNA with the PCR overlapping method; a CMV promoter of the pcDNA3.1 is replaced by the obtained DNA fragment, and the human membrane cofactor mini gene pMCP is constructed. A transgenic mouse built by the invention can change the situation that the ideal animal model is scarce in prior study of human specific pathogen infectious disease, and break through the limits of infectious pathogenesis of corresponding pathogen, vaccine evaluation, drug evaluation and other aspects.

Description

technical field [0001] The present invention relates to the field of pathogenic biology. Background technique [0002] Pathogens can be divided into three categories according to different pathogenic objects, most of which can infect humans as well as animals, and are zoonotic pathogens, such as rabies virus, bird flu virus, etc.; the second category is only Pathogens that infect animals, such as Newcastle disease virus; the third category is pathogens that only infect humans. There are not many types of such pathogens, but they may cause great damage to humans, such as Neisseria gonorrhoeae and Neisseria meningitidis , measles virus, human herpes virus-6, HIV, etc., the losses brought to human society can be said to be well known. Human beings are the only hosts of these pathogens, and there is a lack of effective animal models for their pathogenic mechanisms and control strategies, so they are greatly limited. Some researchers use cell models or tissue culture models to ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/09
Inventor 李国才季明春龚卫娟王劲松朱立天潘兴元王晓红焦红梅
Owner YANGZHOU UNIV
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