Recombinant lentiviral vector containing human osteoprotegerin gene as well as preparation method and application thereof

A technology of recombinant lentivirus and lentiviral vector, which is applied in the field of genetic engineering to achieve the effects of high transduction efficiency, promotion of periodontal bone tissue regeneration, and stable expression

Inactive Publication Date: 2015-05-27
GENERAL HOSPITAL OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of osteoprotegerin in the treatment of periodontal bone defects has not been reported

Method used

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  • Recombinant lentiviral vector containing human osteoprotegerin gene as well as preparation method and application thereof
  • Recombinant lentiviral vector containing human osteoprotegerin gene as well as preparation method and application thereof
  • Recombinant lentiviral vector containing human osteoprotegerin gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 In vitro culture and differentiation of periodontal ligament stem cells

[0082] 1. Primary culture of periodontal ligament cells and detection of physiological indicators

[0083] 1.1 Primary culture of periodontal ligament cells

[0084] The primary culture of cells was carried out by tissue block method and enzymatic tissue block method, respectively. Experimental animals were sacrificed by air embolism. Immediately remove the upper and lower jaws of the rabbit and soak them in 75% alcohol for 15 minutes. In the ultra-clean workbench, the alveolar bone around the teeth was removed with a rongeur, and the rabbit teeth were completely separated. Completely extract the pulp with the pulp-extraction needle, rinse repeatedly from the apex to the crown with PBS containing double antibodies, and cut off the crown and apex with rongeurs, leaving only the middle 1 / 3 of the root, adding 3mg / ml Type I collagenase, digested at 37°C in a water bath shaker for 1 hour...

Embodiment 2

[0099] Example 2 lentiviral vector mediated transfection of periodontal ligament stem cells with hOPG gene

[0100] 1. PCR amplification of human bone protection gene

[0101] Login to Genbank to query the mRNA sequence of human osteoprotegerin gene (Genbank No.NM_002546). According to literature reports, the specific primer sequence of human osteoprotegerin gene cDNA was designed, and the coding sequence region of hOPG gene was synthesized by Invitrogen Company of the United States.

[0102] 2. The construction of the intermediate vector of C-terminal fusion IRES-EGFP

[0103] (1) Design and synthesize 4 primers according to the desired fusion expression gene sequence, the primer sequences are as follows:

[0104] SF-F1:

[0105] 5'-AAATAGATCTGCCACCATGAACAACTTGCT-3' (SEQ NO 1);

[0106] SF-R1:

[0107] 5'-ATACGTCGACAGCTGGGTCTTATAAGCAGC-3' (SEQ NO 2);

[0108] SF-F:

[0109] 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCGCCACCATGAACAACTTGCTGTGCTGCG-3' (SEQ NO 3);

[0110] SF-R2...

Embodiment 3

[0203] Example 3 Lentiviral vector mediated hOPG gene transfection of periodontal ligament stem cells to repair periodontal bone defects

[0204] Rabbit periodontal ligament stem cells were transfected with pLenti6.3-hOPG-IRES-EGFP, compounded with β-tricalcium phosphate (β-TCP), implanted into rabbit periodontal bone defects, and evaluated in vivo The effect of repairing periodontal bone defects in rabbits.

[0205] 1. Isolation and cultivation of rabbit periodontal ligament stem cells, the method is the same as in Example 1.

[0206] 2. Transfect rabbit periodontal ligament stem cells with pLenti6.3-hOPG-IRES-EGFP, the method is the same as that in Example 2.

[0207] 3. Preparation of β-tricalcium phosphate scaffold material Soak granular β-tricalcium phosphate in anhydrous ethanol for 24 hours, rinse and soak in alcohol with a mass concentration of 75% for 15 minutes, rinse with double distilled water for 3 times, and sterilize with high temperature and high pressure Put...

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Abstract

The invention discloses a recombinant lentiviral vector containing human osteoprotegerin gene as well as a preparation method and application thereof. The preparation method comprises the following steps: connecting a human osteoprotegerin gene coding sequence region on a T vector by means of cloning; connecting coding sequence fragments, containing Restriction enzyme cutting sites at two ends, of the human osteoprotegerin gene to a pIRES-EGFP vector by amplification, thereby obtaining an OPG-IRES-EGFP vector; amplifying an OPG-IRES-EGFP sequence, and subcloning to a vector pcDNA6.2w; and recombining the OPG-IRES-EGFP sequence on the pcDNA6.2w to an entry vector via BP, and further recombining to a pLenti6.3/V5 vector via LR, thereby obtaining Lenti6.3-hOPG-IRES-EGFP. The vector can realize sustained and stable expression of transfection gene in body, and can be used for stem cells and primary cells, which are very difficult to transfect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for preparing a recombinant lentiviral vector. More specifically, it relates to a recombinant lentivirus containing human osteoprotegerin gene, a preparation method thereof, and an application of the recombinant lentivirus vector in preparing materials for treating periodontal bone defects. Background technique [0002] Periodontal disease is an inflammatory and destructive disease that occurs in tooth supporting tissues, and is characterized by the absorption and destruction of alveolar bone. In clinical treatment, the basic treatment of periodontal disease, periodontal surgery (root surface treatment technology, guided periodontal tissue regeneration, bone grafting) and drug treatment have a certain effect on eliminating pathogenic factors and reducing inflammation. It is difficult to restore normal alveolar ridge height and form new attachments. [0003] Tissue en...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66C12N15/867C12N7/01C12N5/10A61L27/38A61L27/12A61L27/54
Inventor 苏方刘洪臣
Owner GENERAL HOSPITAL OF PLA
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