جستجوی مقالات مرتبط با کلیدواژه « griffithsin » در نشریات گروه « بیوتکنولوژی و ژنتیک گیاهی »

Atend of 2010 year, about 34 million people living with HIV. With current therapy AIDS is still not curable. Griffithsin (GRFT) is a potent anti-HIV protein that binds to HIV and inhibits cell to cell transmission of virus. In this study, first GRFT gene sequence was optimized. Then pBIgR-KD and pBIgR-ZR vector containing GRFT gene plus N termination of Zera SP ZERA and endoplasmic reticulum retention signal peptide (SEKDEL) was constructed. To obtaining transformants, young leaf explants of Nicotiana tobacco cv. Samsun were transformed with agrobacterium LBA4404 strain containing pBIgR-KD and and pBIgR-ZR. Formed shoots on MS medium containing 1 mg/l BA and 0/1 mg/l NAA and 100 mg/l kanamycin were transferred to root inducing medium (½ MS hormone free) containing 50 mg/l Kn. PCR using GRFT and 35S primers and sothern blot analysis showed succefull integration of GRFT transgene in tobacco genome. RT-PCR and real time PCR showed expression of GRFT gene at transcriptome level with high differences of expression transgenic lines. A band with corresponding size (27 KD for dimer GRFT protein) was detected based on SDS-PAGE analysis. Western blot analysis with anti-GRFT antibody confirmed production of GRFT protein as monomer (13 KD) and dimer (27 KD). Based on ELISA data, targeting to protein bodies using Zera signal peptide resulted in higher amount of recombinant GRFT in comparison to ER targeting via KDEL signal peptide. Based on our results high level of recombinant protein expression is achievable by optimizing of gene codon, selection of suitable host plant and application of suitable promoter and signal peptides.

The production of pharmaceutical compounds having role in treating diseases like AIDS is necessary. In this regard, griffithsin (GRFT) (an algae lectin) can inhibit HIV entry to the cell at picomolar concentration. To production of recombinant GRFT protein, alfalfa, soybean and lettuce were used as hosts for transient expression. Effect of KDEL, signal peptide of carrot extensin and SP subunit of ZERA signal peptide that target protein to endoplasmic reticulum, apoplastic space and space around the cell wall, respectively was studied. 72 hours after agroinfiltration, qRT- PCR analysis showed high potential of lettuce to express of GRFT at transcript level while alfalfa had lowest transcription level. ELISA result using polyclonal anti-mouse anti-GRFT antibody confirmed that targeting of GRFT to apoplast space of soybean led to highest amount of GRFT. Lettuce despite of high level of gene expression, showed low level of GRFT accumulation which may be due to posttranscriptional and post translational modifications. Our results suggest using of soybean leaf tissue for fast and reliable production of pharmaceutical using transient expression.