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Construction method and application of CpG-ODN motif recombinant inhibin plasmid

A construction method and quality technology, applied in the field of biogenetic engineering, can solve problems affecting the development of mammalian follicles, granulosa cells, etc.

Inactive Publication Date: 2018-06-12
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CpG-ODN has shown the ability to improve animal immunity as an adjuvant, it has not been reported whether it affects the development of mammalian follicles, especially the development of granulosa cells

Method used

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  • Construction method and application of CpG-ODN motif recombinant inhibin plasmid
  • Construction method and application of CpG-ODN motif recombinant inhibin plasmid
  • Construction method and application of CpG-ODN motif recombinant inhibin plasmid

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Construction and Identification of Recombinant Inhibitor Plasmid pEGISI-CpG-ODN

[0027] 1. Design and synthesis of CpG-ODN fragments

[0028] Select the CpG motif with activation effect on mouse cells: 5'-GACGTT-3', design the CpG-ODN fragment with immune effect according to the design principles, positive chain: 5'- CTCGAGCGG CACGTT GACGTT CACGTT GACGTT CACGTTGACGTT CACGTT GACGTT CCCAAGCTTGGG -3' (SEQ ID No.1, the underlined part is the cleavage site of restriction endonuclease XhoI and HindIII and their protective bases), synthesized by Huada Gene Technology Co., Ltd.

[0029] 2. Construction and identification of recombinant inhibitory plasmid pEGISI-CpG-ODN

[0030] The CpG-ODN fragment and the pEGISI plasmid with restriction sites were double-digested with Xho I and Hind III restriction enzymes, respectively, and then ligated with T4 DNA ligase overnight at 4°C, and the ligated products were transformed into competent cells E. In coli DH5α, put the ...

Embodiment 2

[0038] Example 2 Discussion on whether CpG-ODN can antagonize the inhibitory effect of inhibin on the development of mouse ovarian granulosa cells

[0039] 1. Isolation and culture of mouse ovarian granulosa cells

[0040] Five 21- to 23-day-old female ICR mice were anesthetized with ether, and quickly killed by decapitation. The mouse abdomen was wiped and disinfected with 75% alcohol cotton balls. The bilateral ovaries were quickly removed in a sterile environment, and redundant fallopian tubes, fat and other tissues were removed under a stereo microscope. Rinse the ovaries with preheated PBS three times, puncture the follicles with a No. 4 needle, and release granulosa cells and oocytes. Add 1 mL of complete medium (DME / F-12+15% FBS+1% penicillin) for pipetting, filter the cell suspension with a 40 μm filter to separate oocytes, and centrifuge the obtained cell suspension at 1000 rpm After 5 minutes, the supernatant was discarded to obtain a cell pellet. Granule cells a...

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Abstract

The invention relates to a construction method of CpG-ODN motif recombinant inhibin plasmid. The construction method comprises the following steps: selecting CpG motif 5'-GACGTT-3' having an activation effect on mice cells, and designing a CpG-ODN fragment having immunity and as shown in SEQ ID NO. 1; and respectively performing the double enzyme digestion on a CpG-ODN fragment and pEGISI plasmid,and connecting, converting and purifying a connection product, thus obtaining the recombinant inhibin plasmid pEGISI-CpG-ODN. After the recombinant inhibin plasmid pEGISI-CpG-ODN transfects mouce ovarian granular cells, the expression of pro-apoptosis genes such as Fas, FasL, DR4 / 5, TRAIL, Caspase8 and Caspase3 mRNA can be extremely significantly reduced, which shows the effect of the CpG-ODN motif resisting the inhibin, the expression of the pro-apoptosis gene is lowered, and the follicular development is influenced.

Description

technical field [0001] The invention relates to biological genetic engineering, in particular to a method for constructing a recombinant inhibitory plasmid containing a CpG-ODN motif, and at the same time relates to the effect of the recombinant inhibitory plasmid on the expression of mouse granulosa cell-related apoptosis genes. Background technique [0002] Mammalian follicle development is a continuous and complex physiological process regulated by various growth factors, pituitary gonadotropins and steroid hormones in the body. During follicular development, inhibin (INH) secreted by granulosa cells is a heterodimeric glycoprotein composed of α and β subunits, which belongs to one of the members of the transforming growth factor β (Transforming growth factor beta, TGFβ) superfamily , regulate the production of steroid hormones and oocyte maturation through autocrine or paracrine, participate in follicular development, and even up-regulate the rate of follicular atresia d...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
CPCC12N5/0682C12N15/85C12N2510/00C12N2800/107
Inventor 王水莲阳美霞李跃伟张虹亮张羽芳
Owner HUNAN AGRICULTURAL UNIV
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