Design, cloning & expression of svsse chimeric gene, a fusion of vegf-a & erbb-2 subunits, for immuno-trap-therapy (itt) of cancer

Mojtaba Koohi leilan,1 Mohammad mir-derikvand,2,*

1. Department of Life Science Engineering, Faculty of New Sciences and Technologies (FNST), University of Tehran, Tehran, Iran
2. Department of Life Science Engineering, Faculty of New Sciences and Technologies (FNST), University of Tehran, Tehran, Iran

Abstract

Introduction

Immuno-therapy is recently considered as an important method in the field of prevention and treatment of cancer. according to oncology studies, most tumor cells have three necrotic, hypoxic and well-oxygenated regions that the hypoxic area prevents immune cell influence and leads to a lack of immune system induction. therefore, cancer immuno-therapy is performed to kill the tumor using synthetic antigens (synthetic immune inducer against cancer) and antibodies. the use of antibody therapy due to its disadvantages, such as the resistance of cancer cells to antibody therapy and the high cost of production, is not an efficient and definitive method. while the direct use of antigens and immune system induction can partly resolve the problems associated with direct antibody therapy, it can be also used to develop the specific cancer vaccines for prevention. in order to make an efficient immune-therapy system, it should be selected an antigen that its malfunction causes the most inhibitory effect on the growth of tumor cells. among the cancer antigens, the family of tyrosine kinase (egfr) and vegf, which are respectively essential factor for metastasis and angiogenesis, can be a good candidate for cancer immuno-trap-therapy (itt). (immuno-trap-therapy: chemotaxis and absorption of immune cells into the entrapped tumor with the aim of identifying and inducing immune system reactions against tumor and cancer cells)

Methods

To design gene construct, after extracting the cdna and the amino acid sequence of the vegf and egfr factors from the gene bank database, the antigenic and joint allelic variants of each subsets of these factors were investigated. immunogenic subunits of vegf-a and erbb-2 variants were selected, and were fused as svsse chimeric gene using the linker (ggsg)7. the sequence of the heat stable ii peptide signal was selected and optimized to secrete this antigen for expression in e. coli hb101 bacteria (the main host for the future). this chimeric construct, svsse, was cloned in pet3a vector after codon optimization for e. coli and then transformed in e. coli expression host bl21-de3.

Results

The svsse gene sequence was confirmed by dna sequencing, double digest (nde1 / bamh1) and pcr. expression conditions of the chimeric gene were determined in the presence of iptg inducer in e. coli bl21-de3 bacteria. the total protein content was extracted using a simultaneous cell lysis and ultrasonic method. ultimately, the expression of the svsse chimeric protein was confirmed using sds-page, his-tag dot blotting and his-tag western blotting.

Conclusion

In this study, in order to investigate the cancer immuno-trap-therapy hypothesis, immunogenic subunit of vegf-a and erbb-2 factors was designed as a chimeric gene and cloned in pet3a vector. the expression of this recombinant gene in e. coli bl21, as the primary bacterium, was indicated by sds-page and blotting techniques. in the future perspective, the recombinant vector will be transformed in the main secretory host (e. coli hb101) and the anticancer performance of svsse chimeric gene and live bacteria will be investigated in the in vivo assessments on rat.

Keywords

Gene cloning, vegf-a, erbb-2, svsse chimeric protein, immuno-trap-therapy (itt), cancer