Construction Of Syncytin Gene Eukaryotic Expression Vectors PCMV-tag 2B-syncytin

Background and objectiveHuman endogenous retroviruses (HERVs) are remnants of germline infections of the human ancestor by retroviruses millions of years ago and occupy about 8% of the human genome. Most of such retroviral genes have lost their coding capacities due to mutations and deletions in evolution. However, some open reading frames have remained by positive selection. Such complete open reading frames express in specific tissues or at specific stages of development, and may play an important physiological role. The aberrant expression of HERVs also has been found in some pathological conditions, suggesting the possible involvement in certain disorders' pathogenesis. HERVs have been indicated the association with a variety of neurological and psychiatric diseases, such as multiple sclerosis (MS) and schizophrenia. Multiple sclerosis associated retrovirus (MSRV) and HERV-K expressions have been detected in the tissue fluid and serum of patients suffered from MS and schizophrenia, respectively. Oluwole detected ERVWE1 env gene expression in affected muscles and unaffected muscles of motor neuron disease (MND) and normal controls with real-time PCR. ERVWE1 env mRNA level was significantly higher in the affected muscles than that of unaffected muscles as well as normal muscles. They also detected SOD1 mRNA levels and found that SOD1 mRNA levels were significantly elevated in the affected muscles as compared with unaffected muscles and normal controls. This suggested per-oxidative impairment in the affected muscles and the association between expression of syncytin and oxidative stress. To our knowledge, this is the first report regarding the aberrant expression of ERVWE1 env in MND tissues. However, the association between aberrant expression of ERVWE1 env and MND has not been clearly determined.In the present study, recombinant plasmid pCMV-tag 2B-syncytin was constructed, and was transfected with liposome into cervical cancer cells Hela, and furthermore, the syncytin mRNA levels were detected by RT-PCR syncytin. The successful construction of recombinant plasmid pCMV-tag 2B-syncytin is important for further exploration of the possible roles and mechanisms of ERVWE1 env gene abnormal expression on motor neuron lesion.Methods①Amplification of the ORF of syncytin gene:total RNA was extracted from human placenta, a pair of primers was designed based on the sequence of human syncytin gene (nucleotide library number:AF072506.2). The whole length of syncytin gene was amplified by RT-PCR. The PCR products were verified by agarose gel electrophoresis.②Construction of eukaryotic expression plasmid pCMV-tag 2B-syncytin:The above PCR product and pCMV-tag 2B vector were digested respectively with restriction enzymes BamH I and EcoRⅠ, and then purified with DNA Fragment Purification Kit. The purified DNA fragments were incubated at room temperature for 5 minutes added with T4 ligase. The ligated products were transformed into Transl-T1 phage resistant chemical competent cells, then inoculated on the Ka+resistance agar dish and the positive clones were picked out.③Identification of the recombinant plasmid:the positive clones, which were determined with PCR, were purified and then digested with restriction endonuclease BamHⅠand EcoRⅠand finally sequenced.④Transfection of human cervical carcinoma cells (Hela):one day prior to transfection, human cervical carcinoma cells were seeded at 3x105cells/well in 6-well plate and cultured in antibodies-free medium. Cells were transfected at about 80% confluence with Lipofectamine 2000 according to the manufacturer recommendation. Cells were transfected with pCMV-tag 2B-syncytin and pCMV-tag 2B, respectively, and were incubated at 37℃and 5%CO2 with medium 1640 containing 10% newborn calf serum.⑤Detection of syncytin mRNA levels in human cervical carcinoma cells (Hela): total cellular RNA was extracted with RNAisoTM Plus reagent according to manufacturer recommendation. Total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase. The syncytin mRNA level was measured with real-time fluorescence quantitative PCR usingβ-actin as an internal control.Result①Successfully cloned the full length of human syncytin coding region from human placenta.②Successfully constructed the eukaryotic expression plasmid pCMV-tag 2B-syncytin. The position, size and orientation of inserted fragments were demonstrated by PCR and restriction endonuclease digestion.③The coincidence between the amplified sequences and the target sequence (AF072506.2) is 99% according to Clustalw analyses. The mismatched bases do not influence the sequence coding and translation.④The results of real time PCR demonstrated that the syncytin mRNA level of Hela cell lines transfected with pCMV-tag 2B-syncytin was significantly higher than that of the cells transfected with empty vector(P<0.01)indicating the successful construction of recombinant plasmid pCMV-tag2B-syncytin, and the effective transfection and expression of the plasmid in human cervical cancer Hela cells.ConclusionRecombinant plasmid pCMV-tag 2B-syncytin has been successful constructed, and this is the basis for further studies of the possible roles and mechanisms of aberrant expression of syncytin in MND.