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Construction method of recombinant adeno-associated virus targeting KLF4 gene silencing

A technology of targeted silencing and construction method, which is applied in the fields of molecular biology and biomedicine to achieve the effects of preventing and treating pulmonary hypertension, inhibiting pulmonary hypertension, and improving the systolic function of the right ventricle

Pending Publication Date: 2019-05-03
AFFILIATED HOSPITAL OF ZUNYI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the application of recombinant adeno-associated virus targeting the KLF4 gene for targeted prevention or treatment of pulmonary arterial hypertension in vivo has not yet been reported in the literature

Method used

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  • Construction method of recombinant adeno-associated virus targeting KLF4 gene silencing
  • Construction method of recombinant adeno-associated virus targeting KLF4 gene silencing
  • Construction method of recombinant adeno-associated virus targeting KLF4 gene silencing

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Construction of recombinant adeno-associated virus targeting silencing of the KLF4 gene:

[0031] 1. Construction of recombinant plasmid pHBAAV-r-KLF4shRNA-GFP

[0032]According to the sequence of the rat KLF4 gene (GenBank Accession Number: NM_053713.1) and referring to the siRNA target sequence selection principle proposed by Tuschl et al., a specific siRNA sequence against KLF4 was designed: CACCCACACTTGTGACTAT. The blank vector (pHBAAV-U6-MCS-CMV-EGFP) was digested with EcoRI and BamHI, and then the siRNA target sequence was inserted into the U6 promoter to regulate its expression. The adenovirus vector contains EGFP gene expression regulated by the CMV promoter. The expression of green fluorescent protein (GFP) is controlled by the CMV promoter, respectively, as a sign of transduction efficiency. Entrusted Shanghai Hanheng Biotechnology Co., Ltd. to synthesize the complete shRNA sequence (annealing of shRNA template, preparation of linearized expression...

Embodiment 2

[0048] Example 2: The preventive effect of recombinant adeno-associated virus rAAV1 expressing KLF4-shRNA on pulmonary hypertension:

[0049] 1. Preparation of pulmonary arterial hypertension rat model, observation of rAAV1-KLF4-shRNA transfection and targeted silencing of pulmonary vascular KLF4 gene:

[0050] Male SD rats were randomly divided into 3 experimental groups exposed to cigarette smoke and 1 healthy control group (Sham) not exposed to smoke. Referring to the method of Jiang X et al. who exposed cigarette smoke for 4 months to construct a pulmonary hypertension model (references Jiang X, Yuan L, Li P, Wang J, Wang P, Zhang L, Sun B, Sun W. Effect of Simvastatinon 5-HT and 5-HTT in a Rat Model of Pulmonary Artery Hypertension. Cell Physiol Biochem. 2015; 37(5):1712-24.). One month before receiving smoke exposure, rAAV1-KLF4-shRNA (1.8×10 11 vg / 200μl / per mouse) the other two groups were injected with equal volumes of rAAV1-control vector (control virus, rAAV1 packa...

Embodiment 3

[0057] Example 3: Therapeutic effect of recombinant adeno-associated virus rAAV1 expressing KLF4-shRNA on pulmonary arterial hypertension

[0058] 1. Targeted silencing effect of rAAV1-KLF4-shRNA injected through the airway on pulmonary vascular KLF4 gene

[0059] Male SD rats were randomly divided into 3 experimental groups exposed to cigarette smoke and 1 healthy control group (Sham) not exposed to smoke. A pulmonary hypertension model was constructed by exposure to cigarette smoke (the method is the same as in Example 2). After 3 months of smoke exposure, a group of rats exposed to cigarette smoke were injected with rAAV1-KLF4-shRNA (1.8×10 11 vg / 200μl / mouse) the other two groups were airway injection of rAAV1-control vector (control virus, rAAV1 packaging irrelevant sequence), and airway injection of normal saline (Saline). After continuing to smoke for 1 month, rats in each group were anesthetized to measure hemodynamic indexes (same as

[0060] Example 2), the rat was...

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Abstract

The invention discloses a construction method of a recombinant adeno-associated virus targeting KLF4 gene silencing. The construction method of the recombinant adeno-associated virus targeting KLF4 gene silencing comprises the following steps: designing a siRNA sequence targeting rat specificity according to a KLF4 gene sequence of the rat; taking pHBAAV-U6-MCS-CMV-EGFP as a blank vector, and constructing a KLF4 interfering adenovirus vector with green-fluorescent-protein (GFP) fluorescence labeling, namely pHBAAV-r-KLF4shRNA-GFP, by adopting an enzyme digestion technology; carrying out sequencing analysis; co-transfecting AAV-293 cells with the recombinant plasmid pHBAAV-r-KLF4shRNA-GFP, a packaging plasmid pAAV-RC and a helper plasmid pHelper; and then, harvesting the recombinant adeno-associated virus rAAV1-KLF4-shRNA. The recombinant adeno-associated virus targeting KLF4 gene silencing constructed by the construction method can be applied in pharmacology for preventing and treatingpulmonary hypertension.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and biomedicine, in particular to a method for constructing a recombinant adeno-associated virus targeting silencing of the KLF4 gene. technical background [0002] Pulmonary hypertension (Pulmonary hypertension, PH) is a common life-threatening disease, characterized by persistent and significant elevated pulmonary artery pressure, which eventually leads to right ventricular failure and death. It is also a key pathological link in the development of many common diseases, such as chronic obstructive pulmonary disease, chronic thromboembolism, and connective tissue diseases. It is an independent risk factor that increases the incidence and reduces survival, and seriously affects the quality of life and prognosis of patients. It deserves our more attention and research. In recent years, although great progress has been made in the study of pulmonary hypertension genes and molecular biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864C12N15/65A01K67/027
Inventor 孙得胜刘虹延欧阳瑶
Owner AFFILIATED HOSPITAL OF ZUNYI UNIV
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