Method for constructing eukaryotic expression vector capable of efficiently expressing pig CYP1A1 genes

A eukaryotic expression vector, -CYP1A1 technology, applied in the field of molecular biology, can solve complex problems such as expression regulation, and achieve remarkable beneficial effects

Inactive Publication Date: 2015-12-09
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, the expression regulation of porcine P450 aromatase is more complicated than that

Method used

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  • Method for constructing eukaryotic expression vector capable of efficiently expressing pig CYP1A1 genes
  • Method for constructing eukaryotic expression vector capable of efficiently expressing pig CYP1A1 genes
  • Method for constructing eukaryotic expression vector capable of efficiently expressing pig CYP1A1 genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction of a eukaryotic expression vector for high-efficiency expression of porcine CYP1A1 gene

[0043] 1. Cloning of porcine CYP1A1 gene

[0044] The experimental pigs were slaughtered Xiaomeishan pigs collected from the Shanghai Meishan Pig Breeding Farm to collect liver tissue samples, extract total RNA, detect RNA purity and concentration by 2.2% formaldehyde denaturing gel electrophoresis and ultraviolet spectrophotometer, and synthesize cDNA by reverse transcription. Primers were designed using the cDNA sequence of the pig CYP1A1 gene in GenBank (NCBIReferenceSequence: NM_214412.1) as a template. In order to facilitate the construction of recombinant vectors, a NotⅠ restriction site (GCGGCCGC) and four protective bases (ATTT) were added to the upstream sequence of the primer, and the downstream The sequence was added with an XhoI restriction site (CTCGAG) and three protective bases (CCG) to form specific primers. Using the reverse transcription cDNA...

Embodiment 2

[0050] Example 2. Verification of the expression effect of pcDNA3.1 / zeo(+)-CYP1A1 eukaryotic expression vector transfected into pig PAM cells

[0051] Prepare 100ug / ml, 150ug / ml, 200ug / ml, 250ug / ml, 300ug / ml Zeocin screening medium (Invitrogen, Carlsbad, CA), and culture PAM cells respectively (see literature: Meng Chunhua, Cao Shaoxian, Su Lei, etc. Isolation, purification and cryopreservation of porcine alveolar macrophages. Jiangsu Agricultural Sciences, 2012, 40(11): 198-20), select the concentration of Zeocin to completely kill PAM cells in 10 to 14 days as the screening concentration of Zeocin (250μg / ml). Normal PAM cells were inoculated in a six-well plate, cultured in complete 1640 medium containing 10% fetal bovine serum and 1% double antibody for 24 hours, and the cell adhesion was observed. When the degree of adhesion reached 80%, transfection reagent Lipofectmine TM In 2000, the pcDNA3.1 / zeo(+)-CYP1A1 plasmid and the pcDNA3.1 / zeo(+) plasmid were transfected into ...

Embodiment 3

[0054] Example 3. Mycoplasma pneumoniae infection experiment of PAM cell line overexpressing porcine CYP1A1 gene

[0055] Use 10 respectively 6 CCU / mL Mycoplasma hyopneumoniae (Mhp) bacterial liquid (see literature: Shao Guoqing, Liu Maojun, Sun Peiyuan et al. Establishment of experimental swine model of swine asthma. Microbiology and Infection, 2007, 2(4): 215-218.) infection adherent 80% CYP1A1 overexpression PAM cells (constructed by the transfection experiment in Example 2 above) (denoted as overexpression group), pcDNA3.1 / zeo (+) vector transfected cells (denoted as empty group), normal For PAM cells (denoted as normal group), 500ml of Mhp bacteria solution and 1000ml of double-antibody-free RPMI1640 culture solution per well. Cells were collected at 0h, 4h, 12h, 24h, and 48h after transfection, total cellular RNA was extracted and reverse-transcribed, and the infection effect was identified by PCR amplification of the specific membrane protein P36 gene of Mycoplasma hyo...

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Abstract

The invention relates to a method for constructing a eukaryotic expression vector capable of efficiently expressing pig CYP1A1 genes and belongs to the field of molecular biology. To clone the CYP1A1 genes in the pig cytochrome P450 family and construct the eukaryotic expression vector, a specific primer containing a specific digestion site and a protective base is designed, a specific coded sequence is obtained through PrimerSTAR high-fidelity enzyme cloning, and a CYP1A1 gene recombination plasmid is constructed, subjected to specific digestion treatment and connected with a linearized vector to obtain the eukaryotic expression vector pcDNA3.1(+)/zeo-CYP1A1 capable of efficiently expressing the pig CYP1A1 genes. The expression vector is used for cell metabolism and specific disease immunoreactions study, the CYP1A1 genes are effectively and excessively expressed under the driving of an efficient enhanser SV40 and an efficient promoter T7PCMV, downstream genes and corresponding regulatory pathways are activated, and cell physiological functions are affected.

Description

1. Technical field [0001] The invention relates to a method for constructing a eukaryotic expression vector for highly expressing pig CYP1A1 gene, belonging to the field of molecular biology. 2. Background technology [0002] The CYP1A1 gene is a member of the cytochrome P450 family, and is the main functional enzyme that metabolizes and activates polyalkylene aromatic hydrocarbons and aromatic amine pro-carcinogens. The latest research shows that cytochrome P450 also plays an important role in the inflammatory response. [0003] At present, most of the reports on CYP1A1 gene focus on the process of human drug metabolism and tumorigenesis. The drug metabolism mechanism of CYP1A1 was explored through enzyme activity, mRNA expression and gene level, revealing that CYP1A1 gene is regulated by aryl hydrocarbon receptors and activates tetrachlorodiphenyl- Pro-carcinogens such as p-dioxin (TCDD) produce highly electrophilic metabolic intermediates, which cause morphological distor...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/79A61K48/00A61P29/00
Inventor 方晓敏任守文徐杰赵为民涂枫王学敏李碧侠付言峰
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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