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pkd2 recombinant overexpression vector and its construction method and use
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An overexpression vector and technology for use, applied in chemical instruments and methods, recombinant DNA technology, botany equipment and methods, etc., can solve problems such as lack of effective treatment and prevention of myocardial infarction
Active Publication Date: 2021-11-30
汉恒生物科技(上海)有限公司
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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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Embodiment 1
[0042] Example 1 Construction of adeno-associated virus vector and transforming embodiment
[0043] 1, pHBAd-EF1-MCS-CMV-EGFP vector (e.g. figure 1 ) With BamHI, EcoRI digestion, digestion system as follows:
[0044]
[0045] 2, the carrier recovery is completed digested.
[0046] 3, PKD2 PCR fragment recovered
[0047] PCR amplification PKD2 sequence system (50 l) as, wherein the forward primer sequence as shown in SEQ ID NO.2,
[0048] Primer sequences inverting NO.3 as shown in SEQ ID.
[0049] Table 2
[0050]
[0051] PCR program: Table 3
[0052]
[0053] The object of the fragments were obtained by gel electrophoresis, such as gel electrophoresis of PCR products wherein figure 2 Indicated.
[0054] 4, the carrier Deal fragment ligation reaction (20μl):
[0055] Table 4
[0056]
[0057] The above reaction system was placed in a hot bath at 50 deg.] C 20min, not immediately after the completion of the reaction as the subsequent reaction sample operations may be tempo...
Embodiment 2
[0063] Injection of recombinant adeno-associated virus vectors myocardial infarction model rats improve symptoms in mice
[0068] Take 24 mice, in which 12 is set in operation between the second and third rib lateral cut of the skin, the chest is exposed. Muscle incision between the second and third ribs, the ribs into the distractor, exposing the heart, left heart fixed exposure atrial, right hand holding the needle holder, with a 8-0 nylon thread 3-5mm LAD at the edge of the appendage ligation, and the other 12 for the sham group, only open chest, not surgery.
[0069] Intravenous injection of 2.2 HBAAV-EF1-PKD2-CMV-EGFP control and adeno-associated virus
[0070] 5 using a needle syringe, injected per mouse 1x10 11 v.g adeno-associated virus, adeno-associated virus control AAV-GFP (green fluorescent p...
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Abstract
The present invention provides a PKD2 recombinant overexpression vector and its construction method and application. The PKD2 recombinant overexpression vector is obtained by inserting the target gene PKD2 into an adeno-associated virus vector, and the adeno-associated virus vector at least includes the following operably linked sequences Elements: EF1 promoter, MCS region, CMV promoter sequence, EGFP gene sequence. The PKD2 recombinant overexpression vector can express PKD2 efficiently. After in-depth research by the inventors, it was unexpectedly found that the PKD2 recombinant overexpression vector can alleviate the symptoms of myocardial infarction in mice. Injecting the adeno-associated virus carrying the gene into the mice with myocardial infarction can effectively restore the heart function of the mice, and the area of myocardial infarction is significantly reduced, indicating that the technical solution of the present invention is suitable for the treatment of myocardial infarction.
Description
Technical field [0001] The present invention relates to a PKD2 recombinant overexpression carrier and its construction methods and use, belonging to the field of biotechnology. Background technique [0002] The gene standard reference sequence of PKD2 is GeneBank NM_000297.3, and PKD2 is located in 4Q21-23, gene length 68KB, contains 15 exons, coding region length 2907 bp. [0003] The prevalence of cardiovascular disease in my country is in the continuous rising stage, with approximately 2 million new intraocular infarction patients, increasing at 10.42% per year, and has become the main cause of heart failure and cardiovascular mortality rise rates and serious hazards One of the difficult diseases of the health of the people. Those skilled in the art have always been committed to the development of technologies that can effectively treat or prevent myocardial infarction. Problems in the prior art are missing methods and methods that can be effectively treated and prevent myocar...
Claims
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