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PKD2 (Polycystic Kidney Disease Gene 2) recombinant overexpression vector and construction method and application thereof

A technology of overexpression vector and construction method, applied in the direction of recombinant DNA technology, chemical instruments and methods, botany equipment and methods, etc., can solve the problems of lack of effective treatment and prevention of myocardial infarction

Active Publication Date: 2019-02-12
汉恒生物科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem in the prior art is the lack of means and methods that can effectively treat and prevent myocardial infarction

Method used

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  • PKD2 (Polycystic Kidney Disease Gene 2) recombinant overexpression vector and construction method and application thereof
  • PKD2 (Polycystic Kidney Disease Gene 2) recombinant overexpression vector and construction method and application thereof
  • PKD2 (Polycystic Kidney Disease Gene 2) recombinant overexpression vector and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Adeno-associated virus vector construction and transformation

[0043] 1. pHBAd-EF1-MCS-CMV-EGFP vector (such as figure 1 ) with BamHI, EcoRI digestion, the digestion system is as follows:

[0044]

[0045] 2. Recover the vector after enzyme digestion.

[0046] 3. PKD2 fragment PCR recovery

[0047] PCR amplification of the PKD2 sequence, the system (50 μl) is, wherein the sequence of the forward primer is shown in SEQ ID NO.2,

[0048] The sequence of the reverse primer is shown in SEQ ID NO.3.

[0049] Table 2

[0050]

[0051] PCR program: Table 3

[0052]

[0053] Gel electrophoresis is used for the obtained target fragments, wherein gel electrophoresis of PCR products is as follows: figure 2 shown.

[0054] 4. Ligation reaction system of the processed target fragment and the vector (20 μl):

[0055] Table 4

[0056]

[0057] The above reaction system was placed in a warm bath at 50°C for 20min. After the reaction is completed, if sub...

Embodiment 2

[0063] Injection of adeno-associated virus recombinant vector into myocardial infarction model mice improves myocardial infarction symptoms in mice

[0064] 1. Experimental materials

[0065] C57BL / 6 mice (available from Shanghai Slack Laboratory Animal Co., Ltd.).

[0066] 2. Experimental method

[0067] 2.1 Modeling of myocardial infarction in mice

[0068] Twenty-four mice were taken, of which 12 were in the operation group, and the skin was cut laterally between the second and third ribs to expose the thoracic cavity. Cut the muscle between the second and third ribs, put in a rib spreader, expose the heart, fix the heart with the left hand, expose the atrial appendage, hold the needle with the right hand, and use 8-0 nylon thread to place 3-5mm LAD at the lower border of the atrial appendage Ligation was performed, and the other 12 animals were in the sham operation group, only the thoracic cavity was opened, and no operation was performed.

[0069] 2.2 Tail vein injec...

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Abstract

The invention provides a PKD2 (Polycystic Kidney Disease Gene 2) recombinant overexpression vector and a construction method and application thereof. The PKD2 recombinant overexpression vector is obtained in a way that the PKD2 is inserted into an adeno-associated virus vector, and the adeno-associated virus vector at least comprises the following sequence elements which can be operably connected:an EF1 promoter, an MCS (Multiple Cloning Site) area, a CMV (cytomegalovirus) promoter sequence and an EGFP (Enhanced Green Fluorescent Protein) gene sequence. The PKD2 recombinant overexpression vector can efficiently express PKD2. An inventor deeply researches and finds that the PKD2 recombinant overexpression vector can alleviate the myocardial infarction symptom of a mouse. The adeno-associated virus vector which carries the gene is injected into the myocardial infarction mouse to effectively recover the heart function of the mouse, and a myocardial infarction area is obviously reduced, which means that the technical scheme of the invention is suitable for myocardial infarction treatment.

Description

technical field [0001] The invention relates to a PKD2 recombinant overexpression vector, a construction method and application thereof, and belongs to the field of biotechnology. Background technique [0002] The gene standard reference sequence of PKD2 is GeneBank NM_000297.3, PKD2 is located in human 4q21-23, the gene is 68kb in length, contains 15 exons, and the coding region is 2907bp in length. [0003] The prevalence of cardiovascular disease in my country is in a continuous rising stage. There are about 2 million new myocardial infarction patients every year, and the rate of increase is 10.42% every year. It is one of the intractable diseases of Chinese people's health. Those skilled in the art have been devoted to developing technologies that can effectively treat or prevent myocardial infarction. The problem in the prior art is the lack of means and methods that can effectively treat and prevent myocardial infarction. SUMMARY OF THE INVENTION [0004] In view of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N5/10C12N15/66A61K48/00A61K38/17A61P9/10
CPCA61K38/1709A61K48/0025A61K48/005A61P9/10C07K14/47C12N15/66C12N15/86C12N2750/14143
Inventor 陈意雄蔡晓龙邹杰朱鹏飞白易鑫
Owner 汉恒生物科技(上海)有限公司
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