| With the sustained deveolpment of the medical treatment, the therapy of cancer is more and more attented, the oncolytic virues provide a new perspective for the treatment of cancer. Newcastle disease virus( NDV) is a member of the oncolytic virues. It shows that the replication of NDV in human tumor cells is 10,000 times more effective than that in normal cells[1]. NDV becomes a potential agent in cancer biotherapy due to its cidal effect on tumor cells in recent reseaches. NDV is an single-stranded non-segmented negative-sense RNA virus, and belongs to paramyxovirus family, paramyxovirus subfamily, avulavirus genus. It mainly infects poultry, it can cause poultry respiratory disease, and it infects human only in rare cases, just a slight conjunctivitis or mild respiratory symptoms and it is a self-limiting disease. It is reported that, NDV is a broad spectrum of oncolytic virus with little damage and slight complications on human body, Since different NDV strains have different antitumor spectrum and their anti-tumor effects are not the same. HN glycoprotein which on the surface of virus, not only mediates the recognition of sialic acid conaining receptors, but possesses neuraminidase activity(NA) which can cleave the sialic acid on those receptors. That is to say both receptor recognition and NA activities possessed by the same protein, it is very important for NDV to possess those two activities so as to bind and to infect. With the sustained improvement of RNA virus reverse genetis techniques of genetic engineering in recent years. People can transform and explore the virus genome on the level of c DNA. NDV HBNU/ LSRC/ F3 strain has been stored in our laboratory, the antitumor effect and mechanism has been confirmed and explored initially, and the whole genome sequence has been sequenced. It belongs to low virulent strains of NDV with strong cleavage site. In order to understand and interpret the antitumor function of NDV F3 better, we choose to study the structure and function of HN protein of NDV F3, It is significant to explore the apoptosis signal pathways of esophageal ECA109 cells induced by NDV F3, so as to grasp the anti-tumor mechanism of NDV F3 better, and to reconst and exert its antitumor properties better.The accession No of HN gene and its protein on the GenBank are KC246549.1 and AGL94562.1. By the use of expert system of protein analysis(ExPASy) provided by ProtParam, protscale, TMpred, Coil, MotifyScan and SWISS-MODEL, etc, and CONSERVE DOMON on the NCBI and SMART, SignalP4.0, Bepipred 1.0 and NetCTL, etc. and Bcepred, ABCpred, SOPMA, SYFPEITHI online bioinformatics analysis program, combined with the PSORT II Prediction, rasmol and other bioinformatics software, so as to analyse and forecast the major characteristic and T / B cell epitopes of the HN protein. The total RNA was extracted from NDV F3, according to the the open reading frame of full-length coding sequence of HN gene(Accession No. KC246549.1) and the multiple cloning sites of the c-flag pcDNA3 vector, the primers were designed with restrinction sites. The HN gene was amplified by the two step reverse transcription PCR, the amplified products were cloned into the multiple cloning sites of the eukaryotic c-flag pcDNA3 vector, c-flag pcDNA3-NDV F3/HN eukaryotic expression system was constructed successfully, the recombinant plasmid was validated by bacterial PCR, double enzymes restriction of the recombinant plasmid and sequencing, The recombinant plasmid c-flag pcDNA3 NDV F3/HN which sequenced exactly right were transfected into ECA109 cells with lipofecter liposomal transfection regent. The expression of protein and the mRNA of NDV F3/HN were tested via the indirect immunofluorescence and RT-PCR, The apoptosis rate of tumor cells transfected by the recombinant plasmid was measured by means of flow cytometry. Since we have demonstrated that caspase-8 has been activated in ECA109 cells infected by NDV F3 in vitro in our previous study, it was suggested that the pathway of death receptor may play a important role in the apoptosis in ECA109 cells induced by NDV F3. In order to study the role of death receptor pathway in ECA109 cells infected by NDV F3 in vitro, we collected the total protein of ECA109 cells once every 4 hour within 36 hours infected by NDV F3, the dynamic expression of cleaved casepase-3, cleaved PARP, FASL, FAS, FADD proteins were detected by western blot. We also observed the effcts of the caspase inhibitor on the apoptosis in ECA109 cells infected by NDV F3 in vitro, so as to observe the effect of caspase dependent apoptosis pathway in ECA109 cells induced by NDV F3 in vitro.The result showed that the RT-PCR product of HN was 1716 bp. Sequence analysis showed that the protein consisted of 571 amino acids, the molecular formula was C2766H4316N752O852S24, The putative protein molecular weight was 62.5kDa, the theoretical isoelectric point was 6.24. HN protein is a transmembrane protein which riched in random curl(Cc) and alpha helix(Hh), and it mainly localized in the mitochondria and cytosol. There were 12 relatively highly hydrophilic region(Parameter score≥1.9), 17 relatively highly surface accessibility region(Parameter score≥1.9), 9 relatively highly flexibility region(Parameter score≥2.0). 36 modification sites of post-translational. 17 potential B-cell epitopes. 12 CTL epitopes and helper T cells combined epitopes. Then it was cloned into the multiple cloning sites of c-flag pcDNA3 eukaryotic expression vector, there was not a mutational nucleobases in the sequencing result compared with the sequence of HN gene of NDV F3 in GenBank; The recombinant plansmids c-flag pcDNA3- F3/HN which has been sequenced exactlly right were transfected into ECA109 cells. The yellow-green fluorescence was observed in cell cytoplasm of recombinat plasmid group 36 hours after transfected by the fluorescence microscope. The mRNA of HN of the recombinat plasmid group was amplified by the reverse transcription PCR in the ECA109 cells 48 hours after transfected, but none in the empty plasmids group, which meaned that the recombinat plasmid was able to express the target protein in enkaryotic cells. We also explored the antitumor properties of c-flag pcDNA3- NDV F3/HN, the apoptosis rate of ECA109 cells in the recomant plasmid group was higher than that in the null vector group 48 hours after transfected. Since the protein of FAS and FASL had been detected in ECA109 cells after NDV F3 infected, we confirmed that the presence of extrinsic apoptotic pathway FAS-FASL-FADD induced by NDV F3. The expression of FADD protein reached the maximum 4 hours after infected, there was a descending trend 4 hours after infected, while there was a rising trend in the expression of activated caspase-3, PARP protein, our previous study showed that caspase-8 and caspase-9 had been activated in ECA109 cells induced by the same concentration of NDV F3. We confirmed that the apoptosis in ECA109 cells induced by NDV F3 were mediated through intrinsic and extrinsic apoptotic pathways together. Since the caspase inhibitor Z-VAD-FMK did not inhibit the cytopathy effect in ECA109 cells induced by NDV F3 completly 24 hours after infected under microscope, it was suggested that the apoptotic pathway of ECA109 cells induced by NDV F3 were mediated through the caspase dependent pathway, and may also through the casepase- independent pathway.The main completed contents of this study are as follow: firstly, we got the main charachters of HN protein of NDV F3, laying the fundation of latter understand the structure and function of HN protein; sceondly, we achieved the expression of HN protein of NDV F3 in eukaryotic expression systerm and explored its antitumor properties preliminarily; thirdly, We confirmed the presence of extrinsic apoptotic pathway of FAS-FASL-FADD induced by NDV F3 and since the caspase inhibitor didn’t inhibite the cytopathic effect of ECA109 cells 24 h after induced by NDV F3, it is probably suggested that there will be caspase-independent pathway induced by NDV F3, which would lay the foundation of fully understanding the antitumor mechanism of NDV F3, thus to reconst and exert its antitumor properties better. |
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