The Establishment And Improvement Of The Sv40 Vector / Into Cos7 Cells And Stable Expression System

As SV40 plasmids replicate out of control in COS7 cells and be lethal to such transfected cells, SV40 vector/COS7 cell system has only been using as transient expression system for a long time. In this research, our original objective is to be able to establish stable expression for SV40 vectors in COS7 cells by introducing a negative feeding-back mechanism into SV40 vector replication procedure. Plasmid pctPA, which is a SV40 expression vector containing SV40 ori, t-PA (tissue-type plasminogen activator) and neomycine gene sequence, is set as a control in experiment. It was anticipated that uncontrolled replication of this plasmid would preclude its foundation of stable transfection in COS7 cells. However, after COS7 cells transfected with pctPA and further selected with G418, significant numbers of drug-resistant colonies arose, which has similar morphology as normal COS7 cells. In these stable, drug-resistant cells, vector pctPA exists as extra-chromosomal episomes with a number of about 300 copies per cell, indicating the vector replicated under the control of cellular regulation system. The expression of t-PA was detected in the culture supernatant of the resistant COS7 cell pool with a level of about 1.1 μg/10~6 cells/day, which can match the yield of vector pctPA in COS7 transient system at the 48th hour after transfection. However, the production rate of t-PA declined sharply following cell passage when there was no G418 existing in culture.The changing trends of the t-PA production rate and episomal copy number were observed for another three months after the formation of stable resistant colonies, which were maintained as a cell pool under half of the previous pressure of G418 (250μg/ml). The yield of t-PA was relatively stable in the first month following cell passage, staying at a level of about 1.1μg/10~6 cells/day. However, the downward trends of t-PA output became obvious after that time, and the expression level dropped to 0.1μg/10~6 cells/day at the 96th day. Southern Blot analysis demonstratedthat similar trend happened to the changes of episomal copy number in the resistant COS7 cells, which showed there were average 262 copies per cell after the first month following cell passage and then the number decreased to 83 copies after the third month. On the contrary, the expression of large T antigen increased in these resistant cells following passage, and gradually closed to the level of that in untrasfected COS7 cells.According to these experimental data, it may be concluded to that a simple SV40 vector (means the plasmid which doesn't contain any regulation element of DNA replication except SV40 ori) can establish stable expression in COS7 cells. Although the production rate decreased gradually following cell passage, the COS7 stable expression system is more simple and need less time to obtain enough recombinant protein when compared with other stable cell systems. With COS7 system, there is no need to choose and identify cell colonies with high expression level and the cells pool can be used for protein production just two weeks after transfection. Moreover, the yields of recombinant protein can be maintained at a relative high level in the following month (>l(j.g/106 cells/day, for t-PA as an example). We think this system may be a good select when many recombinant proteins need to be prepared at same time, for example when screening mutant proteins in protein engineering experiments.In the second part of this research, we found that the co-expression of OBD (or/-binding domain of SV40 large T antigen) could increase the expression of reporter gene (luciferase gene) of SV40 vector in COS7 stable expression system. However, this effect of OBD couldn't be observed when transient COS7, stable CHO or CV1 ceH expression system were used. Southern Blot analysis show that OBD could inhibit replication of SV40 vector in both transiently or stably transfected COS7 cells and result in decreased copy numbers of cellular plasmid, indicating OBD may promotes gene expression through other mechanism. However, when the OBD amino sequence was searched against four major protein signature databasesincluding Pfam, Prints, Blocks and Plofile database, no other sequence profile with statistical significance was found except for on-binding domain of large T antigen. The mechanism of OBD"s effect need to be further studied.In the third part of our research, we found that vector replication and reporter gene expression could be enhanced in stable resistant COS7 cells if exogenous large T antigen of SV40 was provided for the cells, suggesting if the expression of large T antigen could be regulated in cells like COS7, a higher yield of recombinant protein might be achieved by inducing gene expression. So, we designed and constructed an induced expression vector pc4LucpTR3, which containing a reporter gene (luciferase gene) transcription unit regulated by tetracycline (Tet) and a dual-cistron (neomycin and Tet repressor gene) transcription unit, and working as single plasmid style on the basis of T-REx induction system. The expression of luciferase from pc4LucpTR3 increased 8.9 folds when induced by Tet induction in transiently transfected CVl cells. In addition, a cell line SH9, in which the expression of large T antigen can also be regulated by Tet, was established by stable transfection to CVl cells. In theory, the induction effect by Tet on the luciferase expression from vector pc4LucpTR3 in SH9 cells should be stronger than that in CVl cells, as in SH9 cells the expressed TetR from pc4LucpTR3 can inhibit the expression of large T antigen and luciferase gene simultaneously. However, the two induced effects in CVl and HS9 were approximately identical. Southern Blot analysis showed that there was no amplification of episomal DNA in SH9 cells after induced by Tet, indicating the reason for the failure in this experiment might be that the large T antigen or the conditions provided by SH9 cells do not support the replication of SV40 vector.