Past Issue

Volume 9, Supplement 1, Summer 2015 (Presented at 16th Congress on Reproductive Biomedicine and 10th Royan Nursing and Midwifery Seminar) Pages: 96-96

P-128: Optimization of Human LH Gene Expression by Codon Usage Adaptation in CHO Cell Line


Background
Human luteinizing hormone (hLH) belongs to glycoprotein hormones which is composed of two non-covalently linked subunit, α and β. The α-subunit is similar in all glycoprotein hormones, whereas the β-subunit is conferring the hormonal specificity. This hormone has important roles in the growth and maturity of sexual organs and secondary sexual characteristics and steroid biosynthesis in the ovary. LH currently is produced by two methods: isolated from the urine of menopausal women and recombinant technology, but it is not sufficient for medical demands. The purpose of this study is to increase the expression and production of LH. Since each amino acid codon frequencies are not the same in different organisms, replacing codons for each amino acid in the desired gene with the same amino acid codon which is more abundant in the host cells, can increase expression level.
Materials and methods
In this study, open reading frame (ORF) of β subunit was changed based on codon usage in Chinese hamster ovary (CHO). The synthesized gene was purchased in the vector PGEM-B1 and cloned by PCR and inserted in the expression vector pVITRO2-neo-mcs. The a-subunit was already present in the vector. All processes were confirmed by PCR, digestion reaction and sequencing. Recombinant pVITRO2-neo-mcs expression vector was linearized and transfected into the CHO cells. rLH Protein expression was analysed by Bradford's technique, SDS page, Western blotting and ELISA.
Results
Our results showed that According to the standard curve, the levels of intracellular rLH protein was increased but secreted protein was decreased (1.17 and 0.87 folds respectively).
Conclusion
Our results showed that the codon optimizing of LH gene for expression in CHO cell does not change the expression level comparing to the normal gene considerably. It may be because of the LH gene that is a GC rich sequence.