cloning and expression of human vasohibin1 gene in e. coli

Authors

somaye malmir 1 1. biotechnology research center, baqiyatallah university of medical science, tehran , iran.

ali zarei mahmodabadi 1

masoomeh masoomikarimi 2 2. dept. of basic science, torbat heydariyeh university of medical science, torbat heydariyeh, iran.

soheilaalsadat mirhoseini ardakani 3 3. dept. of biology, school of basic sciences, karaj branch, islamic azad university, alborz, iran.

abstract

background: angiogenesis is an important process in various physiologic and pathologic states. the most significant stimulator of angiogenesis is vascular endothelial growth factor (vegf). in contrast, vasohibin1 acts as an angiogenesis inhibitor which specifically inhibits new vessels formation. the aim of the present study was cloning and expression of vasohibin1 gene in e. coli as well as purification of recombinant vasohibin1 protein. methods : total rna was extracted from human umbilical vein endothelial cells and cdna was synthesized by rt-pcr. cdna was amplified using a specific designed primer set. the pcr product was evaluated by electrophoresis and then cloned in pet28a expression vector which transformed into e.coli bl21 (de3) as a host. iptg is used as an expression inducer in media. alternatively, pcr products were analyzed by sequencing and double digestion with ecori and hindiii restriction endonuclease. the expressed protein was purified by ni-nta column and confirmed by sds page and western blotting. evaluation of gene inhibition was carried out through western blottting and rt-pcr. results : no mutation or sequence variants were found in pcr products as a result of sequencing analysis. moreover, the quantity and quality of expressed recombinant protein in the presence of iptg with selected vector in e. coli was approximately high. vash1 significantly prevented the receptor expression. the quality and level of expressed protein in pet28 expression vector indicated the efficacy of the applied system in vasohibin1 production. conclusions : the produced vasohibin1 protein probably can be used as an angiogenesis inhibitor in further studies on retinopathies.

Upgrade to premium to download articles

Sign up to access the full text

Already have an account?login

similar resources

CLONING AND EXPRESSION OF A HUMAN INTERFERON a2 GENE IN E. COLI

The plasmid pALCA1SIFN containing cDNA that encodes the human interferon a-2b was obtained from the ATCC(no. 531667). In this system the expression of the gene is under the control of an alcA promoter. alcA p is a specific promoter for expression of different genes in Aspergillusfilamentous. In this plasmid the coding region of IFN?-2b is preceded by the coding region of a synthetic signal ...

full text

RT-PCR MEDIATED CLONING OF HUMAN GROWTH HORMONE GENE AND I TS EXPRESSION IN E. coli

Human growth hormone (hGH) genomic sequence containing 5 exons and 4 introns was cloned in pcDNA-3 and the constructed plasmid was subsequently used for transfection ofNlli-3T3 cell line using lipofection technique. Expression of hGH in stably transfected cells was assayed using ELISA. Total RNA was extracted from transfected cells and hGH cDNA was amplified by RT-PCR using specific primers...

full text

Cloning and Expression of Bst DNA Polymerase I Gene in E. coli BL21‎

خلاصه DNA پلیمرازها علاوه بر کاربردشان در همسانه سازی و تصحیح ، در انواع تکنیک های ملکولی مانند تکثیر DNA ، جهش های نقطه ای ، توالی یابی DNA ، انواع مختلف PCR ، LAMP و ...اهمیت دارند. پس از کشف PCR تلاش هایی مبنی بر تشخیص و جداسازی آنزیم های مقاوم به دماهای بالا که توانایی تکثیر موثر DNA در دماهای بالا انجام گرفت. در این پژوهش ، سویه Geobacillus stearothermophilus strain 10 به منظور...

full text

Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli

Background: Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals’ foods to hydrolyze phytate and increase absorption of phosphorus. Objectives: Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stabilit...

full text

Codon Optimization, Cloning and Expression of the Human Leukemia Inhibitory Factor (hLIF) in E. coli

Background: Leukemia inhibitor factor (LIF) is a very important pleiotropic cytokine which belongs to interleukin-6 (IL-6) family. LIF exerts multiple effects on different types of cells and tissues with numerous regulatory effects in vivo and in vitro. It is a lymphoid factor, which performs a number of activities including cholinergic neuron differentia‌tion, contro...

full text

Cloning and Expression of Human Gamma-Interferon cDNA in E. coli

Prior to the production of human gamma interferon using recombinant DNA technology, it had been producedmainly upon mitogenic induction of lymphocytes in very low amounts, which evidently hamperedits characterization and its medical applications. The recombinant gamma interferons produced in largerquantities in prokaryotic systems retain their biological activities, and can be...

full text

My Resources

Save resource for easier access later


Journal title:
international journal of health studies

جلد ۳، شماره ۱۱، صفحات ۰-۰

Hosted on Doprax cloud platform doprax.com

copyright © 2015-2023