PTEN (phosphatase and tensin homolog) gene interference shRNA3, and recombinant adenovirus vector and construction thereof

A technology of recombinant adenovirus and gene interference, which is applied in the field of animal molecular biology and genetic engineering, can solve the problems of gene mutation and carcinogenesis, and achieve the effect of promoting the accumulation of fat

Active Publication Date: 2014-12-10
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of the lentiviral vector delivery system are similar to those of the retrovirus. It can also integrate the transferred exogenous gene into the host cell genome, which has certain risks to the gene expression of the host cell and may cause gene mutation and canceration. Wait

Method used

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  • PTEN (phosphatase and tensin homolog) gene interference shRNA3, and recombinant adenovirus vector and construction thereof
  • PTEN (phosphatase and tensin homolog) gene interference shRNA3, and recombinant adenovirus vector and construction thereof
  • PTEN (phosphatase and tensin homolog) gene interference shRNA3, and recombinant adenovirus vector and construction thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] This example illustrates the design, synthesis and screening of PTEN gene shRNA.

[0044] According to the experimental principle of RNAi, four pshRNAs were designed and synthesized according to the PTEN gene, named pshRNA1 (SEQ ID No.1), pshRNA2 (SEQ ID No.2), pshRNA3 (SEQ ID No.3), pshRNA4 (SEQ ID No.4), and these fragments were chemically modified with green fluorescence to facilitate the observation of the transfection effect after transfection. Then connect the four fragments to the carrier pGPU6 / GFP / Neo respectively.

[0045] On the basis of using the improved collagenase two-step perfusion method to separate calf hepatocytes, low-speed centrifugation and adding erythrocyte lysate were used to reduce the number of red blood cells and improve the purity of hepatocytes. After cell counting, 2 mL of cell suspension was inoculated into each well of the six-well culture plate, so that the final inoculation density was 1 × 10 5 piece / cm 2 . After 4 hours, the adhere...

Embodiment 2

[0050] This example illustrates the construction of a recombinant shuttle vector.

[0051] Digest the pShuttle-Basic-EGFP recombinant shuttle vector with endonuclease KpnI / BamHI, cut the gel after 1% gel electrophoresis, and recover the target gene fragment of about 4.2Kb vector, because prolonged ultraviolet irradiation may damage DNA, so this step Electropherograms are not stored. The specific enzyme digestion system and reaction conditions are shown in Table 1.

[0052] Table 1 Shuttle plasmid digestion system and reaction conditions

[0053]

[0054] The original plasmid pGPU6 / GFP / Neo-PTEN-shRNA3 was double digested with KpnI / BamHI, after 1% gel electrophoresis, the target fragment was cut out under ultraviolet light, and the DNA gel recovery and purification kit (column centrifugal type) was used. (Refer to the instructions of the kit for specific steps, which are omitted here) Steps such as recovery and purification, the target gene fragment (U6+PTEN-shRNA3) of abou...

Embodiment 3

[0061] This example is used to illustrate the enzyme digestion identification of the recombinant shuttle plasmid.

[0062] Use KpnI / BamHI (interfering recombination shuttle plasmid) endonuclease to identify the recombinant shuttle plasmid, and the expected results are: negative clone of interference recombination shuttle plasmid: a band of 4.2 kb, and positive insert clones are 0.3 kb and 4.2 kb Kb two bands. The enzyme digestion identification system and reaction conditions are shown in Table 4. After enzyme digestion, 1% agarose gel electrophoresis was taken, and the results were as follows: figure 2 shown.

[0063] Table 4 KpnI / BamHI enzyme digestion recombinant shuttle plasmid enzyme digestion identification system and reaction conditions

[0064]

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Abstract

The invention relates to a PTEN (phosphatase and tensin homolog) gene interference shRNA3 of which the nucleotide sequence is shown in SEQ ID No.1. The invention also provides a recombinant adenovirus vector containing the PTEN gene interference shRNA3 as well as a construction method and application thereof. According to the invention, by selecting an RNAi (RNA interfere) technology, a pAdxsi-GFP-PTEN-shRNA vector is successfully constructed, and through culturing and amplifying, an interference recombinant adenovirus with a titer of 1.2*10<10> PFU (Plaque Forming Unit) / ml is obtained, so that the expression of PTEN genes can be reduced, and due to the low expression of PTEN, the assembling and secretion of VLDL (very low-density lipoprotein) are reduced, and then the accumulation of intracellular TG is caused, thereby promoting accumulation of fats in liver cells.

Description

technical field [0001] The invention belongs to the field of animal molecular biology and genetic engineering, and relates to a PTEN gene interference shRNA3 and a recombinant adenovirus vector and its construction. Background technique [0002] Fatty liver of dairy cows during the peripartum period is a metabolic disease caused by the negative energy balance of dairy cows during the transition period. However, due to the lack of effective control measures, it has brought huge losses to the cattle industry, and it is urgent to find an effective treatment method. In view of the weak liver fatty acid oxidation ability of peripartum dairy cows (especially Holstein cows), the insufficient ability to synthesize and secrete lipoproteins is the main link in the occurrence of fatty liver, and the establishment of gene therapy to regulate liver fatty acid oxidation and VLDL secretion and synthesis may be possible. It can provide new ideas for the prevention and treatment of fatty liv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/861C12N15/66A61K48/00A61P1/16
Inventor 付世新罗春海贺显晶沈冰蕾郭景茹
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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