Production of HBsAg100-GST recombinant protein as an immunogen model for generating antibody in mice

Abstract

Since years ago, a new paradigm of vaccine design is emerging. Instead of attenuated virulent microorganisms or killed virulent microorganisms, effective subunit vaccines were developed using recombinant DNA technology. Biosynthesis of recombinant protein in Escherichia coli may offer an alternative procedure to generate therapeutic protein free from human protein. In this research, hydrophilic domain of S protein (aa 100-164)-encoding gene of hepatitis B surface antigen was cloned for vaccine candidate production. The gene was ligated with pGEX-4T-2 vector and sequenced. Sequences alignment of the amplified fragment with genome of hepatitis B virus indicated that the sequences were identical. In this research, cloned DNA fragment of Hepatitis B surface antigen was placed downstream from the gluthatione S-transferase (GST) protein-encoding gene in expression plasmid pGEX-4T-2 and expressed in Escherichia coli cells. A polypeptide of 34.8 kDa molecular weight was synthesized and identified as HBsAg100-GST fusion proteins. The recombinant proteins were then purified using GSTrap and HiTrap column and could be used for vaccine candidate or for antibody generation. The purified protein was tried to trigger cell immune to produce antibody in mice. Results indicated that the immunogenicity of HBsAg100-GST was higher than GST protein in elicit the levels of HBsAg100-specific IgG antibody in mice. These results suggest that the HBsAg100 produced in E. coli has immunogenicity. A major result achieved from this research was clones carrying S antigens-encoding gene that could be used further for production of recombinant hepatitis B vaccine candidates.

Kemajuan teknologi molekuler dalam beberapa dekade terakhir, terutama sejak ditemukannya sekuen genom lengkap dari mikroba-mikroba patogen, telah menemukan jalan baru bagi dihasilkannya berbagai jenis protein rekombinan, baik vaksin, antibodi, maupun peptide sintetik yang memiliki manfaat tertentu. Pada saat ini, vaksin telah dihasilkan dengan teknologi DNA rekombinan, yaitu melalui kloning gen penyandi protein tertentu pada mikroorganisme patogen yang dilanjutkan dengan ekspresi gen tersebut pada sel hewan, sel tanaman, ataupun pada bakteri. Penggunaan mikroorganisme virulen yang dilemahkan ataupun yang dimatikan telah diganti dengan penggunaan vaksin sub unit yang lebih efektif dengan teknologi DNA rekombinan. Melalui penggunaan teknologi tersebut, gen tertentu dari mikroorganisme virulen dapat dikloning, diekspresi dan dievaluasi penggunaannya sebagai vaksin. Tersedianya bioteknologi rekayasa genetika yang dilahirkan pada tahun 1973, telah memungkinkan manusia untuk mengisolasi gen (serangkaian molekul DNA) serta memanipulasinya, kemudian memindahkan gen tersebut dari satu organisme ke organisme lain. Peranan bioteknologi dirasakan semakin bertambah besar dalam menunjang kegiatan pembangunan industri di berbagai sektor, terutama sektor kesehatan dan pertanian termasuk sub sektor peternakan.