Construction method and application of human hysteromyoma 14-3-3 gamma high expression vector

A technology for uterine fibroids and expression vectors, which is applied in the field of construction of human uterine fibroids 14-3-3 γ expression vectors, can solve the problem of lack of objective basis for the mechanism of action, and achieve convenient screening, simple and effective preparation process, recombinant stable effect

Active Publication Date: 2014-02-05
THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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Problems solved by technology

[0004]In our previous study, we took the lead in using two-dimensional gel electrophoresis and electrospray tandem mass spectrometry to study the differential proteins between uterine leiomyoma and surrounding normal muscle tissue, and adopted Further verification by RT-PCR and Western blot found that the expression of 14-3-3γ in uterine fibroids was significantly lower than that in normal myometrial tissue, but the specific mechanism of 14-3-3γ in the development of uterine fibroids is still lacking objective basis

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  • Construction method and application of human hysteromyoma 14-3-3 gamma high expression vector
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  • Construction method and application of human hysteromyoma 14-3-3 gamma high expression vector

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Embodiment Construction

[0030] Such as figure 1 As shown, the construction method of the human uterine fibroid expression vector of the present invention is selected pCMV-N-Flag As an intermediate carrier, design a pair of primers, introduce BamH I and EcoR I enzyme cutting sites into the primers, use the RNA containing the target gene as a template for PCR amplification after reverse transcription, cut off the target band, and recover it with the kit Purification, the purified PCR product and vector pCMV-N-Flag After double digestion with BamH I and EcoR I, use the kit to recover and purify the digested product, and then connect the fragments, screen and culture, and clone to obtain the human uterine leiomyoma expression vector.

[0031] The following are construction examples and applications of specific expression vectors:

[0032] 1. Target gene preparation

[0033] (one) RNA extract

[0034] The main components in TRIzol reagent are guanidine isothiocyanate and phenol, among which gua...

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Abstract

The present invention discloses a construction method and an application of a human hysteromyoma 14-3-3 gamma high expression vector. The construction method comprises: amplifying and purifying target gene, designing an upstream primer and a downstream primer, extracting hysteromyoma RNA, carrying out reverse transcription to obtain a template, carrying out PCR amplification, cutting the target band, carrying out recovery and purification with a kit, constructing and identifying recombinant plasmid 14-3-3gamma-pCMV-N-Flag, carrying out double digestion on the purified PCR product and the vector pCMV-N-Flag with BamHI and EcoRI, respectively adopting kits to recover and purify the digestion products, linking the fragments, carrying out screening culture, cloning, identifying, sequencing, carrying out WesternBlot to detect protein expression, adopting CCK-8 to detect cell proliferation change, and adopting a AnnexinV-FITC/PI double staining method to detect apoptosis through flow cytometry.

Description

Technical field [0001] The present invention involves the construction method and application of a human uterine fibroids 14-3-3 γ expression vector. Background technique [0002] Uterine fibroids are one of the most common tumors in the human body and the most common tumor in female reproductive organs.According to statistics, the incidence of uterine smooth muscle tumors in women aged 30 to 50 is as high as 20%to 30%.In recent years, its prevalence has increased year by year.Although uterine fibroids are benign tumors, it is the first cause of uterine resection of women in fertility, which seriously endangers women's reproductive health.The occurrence of uterine fibroids is closely related to estrogen and progesterone, but the molecular mechanism of its development is not clear.At present, drugs used to treat uterine fibroids mainly include gonadotropin to release hormone analogs (GNRHA) and Mifei, but there are risk of increasing reflux of uterine fibroids after drug discontin...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/66
Inventor 朱雪琼沈奇姜文晓
Owner THE SECOND HOSPITAL AFFILIATED TO WENZHOU MEDICAL COLLEGE
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