Construction Of Recombinant Plasmid PcDNA3(-)SVT And Its Expression In Intestinal Epithelial Cells Of Mouse

Small intestine is the main organ for digestion and absorption of nutrients. Intestinal epithelial cells (IEC) are the primary functional cells in intestinal tract. Besides participating in digestion, absorption, immunization barrier and stress reaction, IEC plays a vital role in internal and external secretory. Most researchers mainly use primary cultured cells for their studies, which cost lots of time and money and result in relative complex preparation steps in manipulation and in common variations in cell physiology. Consequently, it is difficult to compare experiments while using different passages of cells. Immortalised cell lines provide more advantages than primary cells, such as uniformity, availability and easier genetic manipulation. Create an immortalistic IEC lines is one of the major means to study intestinal function and regulatory mechanism of pathology, cell differentiation and nutrients absorption.The objective of this research was to design and construct eukaryotic expression vector of pcDNA3(-)SVT. pcDNA3 was digested with PVUII and reconstructed a plasmid named pcDNA3(-). pGEM-LT was digested with XholI to obtain the SV40 large T antigen gene fragment. The target fragment was insert into eukaryotic expression vector pcDNA3(-) and confirmed by gene sequence analysis.To obtain pure IEC, an experiment was carried out to purify mouse intestinal epithelial cells by means of isolation, passaging and purification. Tissues were collected from fetal mouse intestine and the approach of tissue plot culture was conducted, the primary cells were purified through scraping, two step digestion. For passaging, 0.05% Trypsin-EDTA was used. The IEC was validated by morphological observation and immunostaining.The reconstructed vector was transfected into mouse IEC by neans of Lipofectin transfection method. At 48 h after transfection, the expression of SV40 T was detected by either PCR or RT-PCR with specific primers of large T gene respectively. The genome DNA and total RNA were isolated from the positive cells. The results showed that: the sequence was consistent with the nucleotide sequence of gene SV40 large T antigen gene published, as well as the inserted location and direction of fragment, which indicated that the eukaryotic expression recombinant vector pcDNA3(-)SVT was constructed. The recombinant plasmid pcDNA3(-)SVT could be a stable and valuable molecular tool for eukaryocyte study. Through tissue culture, the growth of cells was in good condition and we can obtain more pure mouse intestinal cells after purification. And the cells were confirmed by immunohistochemistry. The mouse IEC provided the cell material for the study of cell immortalization. With these samples, the specific 372 bp fragment was amplified using PCR and RT-PCR, which speculated that the simian virus 40 large T antigen gene was transfected stably into mouse IEC and expressed positively. This preliminary study contributed foundation for the establishment of mouse immortalized intestinal epithelial cell line.