UNAIDS has estimated 33.3 million people living with HIV at 2009. By HAART therapy, AIDS is not longer heritable, but it's still not curable or preventable. Griffithsin (GRFT) is a novel anti-HIV protein from the red alga Griffithsia. The ability of GRFT to restrict HIV entry into cells at a trillionth of a gram made it more potent than other inhibitors. GRFT bound to viral coat glycoproteins (gp120, gp41, and gp160) and block binding of virus to CD4 receptors in cell. Engineering of E. coli to produce GRFT did not yield large enough quantities for use as a tropical microbicide. Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in single transformation event and transgene containment by maternal inheritance. GRFT gene sequence (Gene.Bank. CS255217.1) was taken from NCBI. Desired primers were designed by Primer 3 and restriction sites in gene codon identify using BIOlabs-NEB Cutter. Codon optimization was carried out to ensure high expression of gene. Some part of gene changed according to lettuce chloroplast genome and suitable restriction sites designed. PCL 96–39 chloroplast transformation vector and synthesized gene digested with SalΙ and HindIII. Then fragment recovery, ligation and transformation to E. coli (DH5α strain) were carried out. After obtaining recombinant colonies on selectable medium containing ampicillin, testing of colonies by double digestion and PCR confirmed accurate integration of 500 bp fragment to the vector. Lettuce cotyledon bombarded with this new construct containing GRFT gene. In future, we will test integration of GRFT to plastome of lettuce this valuable protein in high level.
