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Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof

A technology of recombinant plasmids and gene vaccines, applied in biochemical equipment and methods, botany equipment and methods, gene therapy, etc., can solve the problems of mouse embryo death, embryonic dysplasia, etc., and achieve the reduction of mouse fertility and strong immune effect , the effect of stable operation

Inactive Publication Date: 2011-09-21
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is also reported that excessive intake of RA during pregnancy in mice may lead to embryonic dysplasia, the type and degree of malformation are dose-related, and loss of Cyp26a1 function can cause mouse embryonic death

Method used

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  • Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof
  • Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof
  • Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 : Cloning the full-length cDNA of rat uterus Cyp26a1

[0026] This embodiment is the full-length cDNA clone of rat uterus Cyp26a1, and the specific steps are as follows:

[0027] (1) Animal tissue collection: Select sexually mature Spreqne-Dawley female rats weighing 220-260 g, and mated with male rats (1:1) in the estrus period, and take pregnant D3-D6 uteri, and remove the mesentery tissues and embryos.

[0028] (2) Extraction and purification of total RNA from the pregnant uterus: using RNAgents from Promega The Total RNA Isolation System kit was used to extract the total RNA of the pregnant uterus, and the TRIzol was used to extract the total RNA of the tissue in one step. Cut the tissue with bacterial scissors, transfer the homogenate to an Eppendorf tube, add 0.2ml chloroform, shake for 15 sec, let stand at room temperature for 2-3min, centrifuge at 12,000rpm for 15min (4°C), take the supernatant to a new Eppendorf tube, Add 0.5ml of isopropanol, mi...

Embodiment 2

[0036] Example 2 : Construction of recombinant plasmids containing Cyp26a1

[0037] This example is the construction of the recombinant plasmid containing Cyp26a1 of the present invention, which is to recombine the full-length cDNA fragment of Cyp26a1 into PCR 3.1 In the eukaryotic expression plasmid (Invitrogen product, purchased from Beijing Zhongyuan Leading Technology Co., Ltd.), see the specific plasmid map figure 2 , the recombinant plasmid constructed is the anti-fertility PCR3.1-Cyp26a1 gene vaccine, and the specific operations are as follows:

[0038] The PCR product Cyp26a1 cDNA fragment amplified after purification and recovery in Example 1 was connected into the pMD18-T vector (Takara product, purchased from Beijing Liuhetong Economic and Trade Co., Ltd.) and transformed into DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd. Ltd.). Pick the positive clone and shake the bacteria, and after the sequencing and identification are cor...

Embodiment 3

[0039] Example 3 : In vitro expression identification of pCR3.1-Cyp26a1 gene vaccine

[0040] This example is for the pCR3.1-Cyp26a1 gene vaccine constructed in Example 2, to carry out the identification of the in vitro transient expression and expression content of the exogenous Cyp26a1 gene, which is divided into two parts: the in vitro mRNA expression of the Cyp26a1 gene and the in vitro protein expression of the exogenous Cyp26a1 .

[0041] (1) In vitro mRNA expression of Cyp26a1 gene:

[0042] The in vitro transient expression system of human cervical cancer (HeLa) cells and Chinese hamster ovary (CHO) cells (purchased from the Cell Center of the Basic Institute of Peking Union Medical College) was established. The specific operations are as follows: PCR3.1-Cyp26a1 eukaryotic expression plasmid was injected into After transfection, the culture medium was collected at 24 hours, 48 ​​hours, and 72 hours, and the total RNA of the collected transfected HeLa cells and CHO c...

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Abstract

The invention provides an antifertility recombinant plasmid and genetic vaccine, and a preparation method and applications thereof. The recombinant plasmid consists of Cyp26a1 gene cDNA fragment and eukaryotic expression vector pCR 3.1. The preparation method of the recombinant plasmid and genetic vaccine comprises the following steps: extracting and purifying the total RNA of rat pregnant uterus, designing a specific primer, adopting the temperature reducing reverse transcription-polymerase chain amplification method to clone and amplify the Cyp26a1 gene cDNA fragment and recombining the fragment in eukaryotic expression plasmid. The prepared genetic vaccine can be used to inhibit the fertility of mammals and prevent and / or control rats, have the advantages of safety, long-term effectiveness, stability and convenient operation and have higher immune effect than other immune means; and the genetic vaccine can be used to effectively reduce the fertility of rats and obviously reduce newborn offspring rats.

Description

technical field [0001] The invention belongs to the technical field of biology and modern agriculture, and relates to an anti-fertility gene vaccine, more specifically, the invention relates to an anti-fertility gene vaccine containing rat cytochrome P450 family 26 superfamily A polypeptide 1 (Cyp26a1), Its preparation method and use. Background technique [0002] Rats and other small harmful mammals cause serious losses to global agriculture, forestry and animal husbandry every year. The average annual loss of 2106 tons of grain is caused by the rat infestation of the house mouse (Mus domesticus) in the grain-producing areas of southeastern Australia. In the 1980s, rodent infestation in my country's agriculture, animal husbandry and forestry industry generally occurred. Every year, the area of ​​rodent infestation in agricultural and pastoral areas reached tens of millions of hectares, reducing more than 10 billion kilograms of grain, tens of billions of kilograms of pastu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N1/21C12N5/10A61K48/00A61P15/18
Inventor 彭景楩夏红飞韩丙辰孙敬杨颖
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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