Constructing method of bicistronic recombinant adenovirus expressing neurotrophic factor and tyrosine hydroxylase

A tyrosine hydroxylase bicistronic, neurotrophic factor technology, applied in the field of genetic engineering, can solve the problems of backwardness in superiority, and the construction of bicistronic vector Parkinson's disease and other problems

Active Publication Date: 2012-11-07
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with foreign countries, the types of vectors used in China or the superiority of vectors are far behind, and bicistronic vectors have not yet been constructed to treat Parkinson's disease

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Constructing method of bicistronic recombinant adenovirus expressing neurotrophic factor and tyrosine hydroxylase
  • Constructing method of bicistronic recombinant adenovirus expressing neurotrophic factor and tyrosine hydroxylase
  • Constructing method of bicistronic recombinant adenovirus expressing neurotrophic factor and tyrosine hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0034] (1) Construction and packaging of bicistronic recombinant adenovirus

[0035] Insert the NTN gene into the multi-cloning site B of the bicistronic expression plasmid pcDNA3.0BA by using the EcoRV and Xhol restriction sites, screen the recombinant plasmid pcDNA3.0BA-NTN, and then insert the TH gene through the BamHI and EcoRI restriction sites. Insert the plasmid into the multiple cloning site A, screen the recombinant plasmid pcDNA3.0BA-NTN-TH, use the recombinant plasmid as a template, P1: 5'-TTGTCGACACCATGTCCATGTTGTTCTACAC-3' as the upstream primer, P2: 5'-GGGATATCTTAGCCAATGGCACTCAGCGC- 3' is the downstream primer, and two restriction sites, SalI and EcoRV, are introduced into the bicistronic expression cassette for PCR amplification, see figure 1 , figure 2 .

[0036] The PCR amplified product of the bicistronic expression cassette was recovered by gel cutting and inserted into the adenovirus shuttle plasmid pShuttleCMV, because the upstream and downstream multipl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a constructing method of bicistronic recombinant adenovirus expressing neurotrophic factor and tyrosine hydroxylase (TH) and belongs to the genetic engineering. The method comprises the following steps: NTN (Nao Tai Nutrient) and TH genes are inserted in the polylinker of bicistronic expression plasmid pcDNA3.0BA respectively, the screened template and primer are utilized to perform PCR amplification and obtain a bicistronic expression cassette, the expression cassette is inserted in the adenoviral shuttle plasmid pShuttleCMV to transcribe and translate, competent cellsperform electricity facing, positive clones are screened, XL-10(Gold) host bacteria are identified and transformed, and the bicistronic recombinant adenovirus is packaged in AD-293 cells. After the host cells are infected by the recombinant adenovirus Ad-NTN-TH, the adenovirus can transcribe and express both NTN and TH genes stably; the expressed functional protein NTN promotes the chick-embryo dorsal root ganglion to grow synapses in vitro and can also promote the survival of fetal rat dopaminergic neurons; and TH is utilized to catalyze the reaction of L-Tyrosine in vitro and generate L-DOPA.

Description

technical field [0001] The invention belongs to genetic engineering, in particular to a method for constructing a bicistronic recombinant adenovirus expressing neurotrophic factors and tyrosine hydroxylase. Background technique [0002] Parkinson's disease is a degenerative disease of the central nervous system, characterized by degeneration and necrosis of dopaminergic neurons in the substantia nigra of the midbrain and decreased synthesis of dopamine (DA). Main clinical manifestations: resting tremor, bradykinesia, muscle rigidity, abnormal posture and gait, and most patients died of complications caused by lung infection. Parkinson's disease mainly occurs after the age of 60. With the increase of the aging population, the number of patients with Parkinson's disease and the resulting economic burden on the country and society will be huge in the next 10-15 years . There are as many as 4 million sick people in the world, and about half of them are in our country. The mos...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N15/861
Inventor李鸿钧孙茂盛王文举严敏周艳
OwnerINST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI