Leishmaniasis is a major infectious disease of considerable public health in more than 86 countries around the world. Several approaches toward vaccine development against this disease have been taken. Glycoprotein (gp63) is conserved among diverse species of Leishmania and has induced immunological responses in murine models. Therefore, this glycoprotein has been considered as a second generation vaccine for Leishmaniasis and for potential diagnostic antigen. Recombinant vaccine using gp63 in cocktail form is one of the candidates. Since, Pichia pastoris expression system is similar to that of the eukaryotic genes, refolding and glycosylation aspects of the expressed protein, gp63 gene from NIH strain of L. major cloned into BamHI site of pHIL-S1 as yeast expression vector (shuttle vector). This vector carries sequences of acid phosphatase (PHO1) signal peptide from yeast. The construction transfected into the P. pastoris using lithium chloride method. Recombinant clones were screened on histidin minus media. The expression of rgp63 was studied by methanol after induction. The expressed recombinant protein was confirmed by Western blotting and electron microscopy. The expression level of rgp63 was more than 30%. Since the rgp63 expression in P. pastoris was active in SDS-PAGE gelatin gel , therefore it should be very similar to the native form

Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for target-ing Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. Methods: Four single guide RNAs were designed to target sequences in the coding re-gion of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2. Results: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA frag-ment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was de-tected in about 50% of transfected cells. Conclusion: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engi-neering of cells and model animals.