Apoptosis Induced By VP1of Foot-and-mouth Disease Viurs In Calf Thyroid Cells And The Conforming Of It’s Functinal Domain

[Objective] To find the effective domain of Foot-and-mouth disease virus(FMDV) VP1inducing apoptosis on CTY, the primary calf thyroid cells (primarycalf thyroid, CTY) was prepared and cultured, and the VP1and it’s truncatedmutation was transfected into CTY.[Method] Acording to before studies, the CTY was prepared with improvement.The sequences of primers was designed based on the published sequence of FMDVVP1with FLAG tag and the full lenghth VP1gene of FMDV was amplified byRT-PCR and subcloned into expression vector pCAGGS by restriction endonucleaseEcoR I and Xho I, and the recombinant plasmid was confirmed by sequencing andrestriction endonuclease digestion. The VP1protein’s expression is analyzed byWestern-Blot after Lipofectamine2000transfection and the apoptosis induced byFMDV VP1protein in primary calf thyroid cell was detected through observing underthe microscope. And the FMDV VP1protein induced CTY apoptosis verified byAV-PI staining, mitochondrial membrane potential change and Hoechst-33258fluorescent staining, and the empty vector pCAGGS seemed as negtive control. Thetruncated mutation of FMDV VP1was constructed to find the effective domain ofFMDV VP1inducing apoptosis on CTY. The specific program was as follow:VP1-1(1-213)、VP1-2(181-360)、VP1-3(331-510) VP1-4(481-639). The recombinantplasmid and pCAGGS transfected respectively by Lipofectamine2000and the rusultdetected by flow cytometry with AV-PI staining.[Results] The result we obtained as follow:1. The CTY was prepared and it was quickly attached to the wall of the bottleobserved by microscope. The CTY began to propagated after30h and formed cellisland.72h later, the CTY became compact monolayer cells and transparent like daisypetals. The cell morphology was normal and could be used for further study with clearboundaries and formed multiple layers.2.The desired gene was cloned with a appropriate size and subcloned to expression vector pCAGGS by restriction endonuclease EcoR I and Xho I. Therecombined plasmid was obtained two fragments which is5000bp and600bprespectively digested by EcoR I and Xho I and the sequence is exactly.3.The analysis of Western-Blot demonstrated that FMDV VP1protein can bewell expressed in CTY, whereas the negtive control group transfected with emptyvector pCAGGS couldn’t. In addition, the CTY cells transfected with the plasmidpCAGGS-VP1was observed irregular in shape and majority cells was shedding,whereas the negtive control group transfected with empty vector pCAGGS was not.The apoptosis induced by FMDV VP1protein in primary calf thyroid cell by AV-PIstaining was serious and the result is agreed after transfected24h and48h. Meanwhile,the experimental group transfected with pCAGGS-VP1had fluorescence under thefluorescence microscope, but the control group had not, which revealed that FMDVVP1could induce apoptosis in CTY. Using JC-1as fluorescent probe to testVP1induced apoptosis in CTY found that after transfected24h and48h respectively,the exprimental group had a2-3higher apoptosis rate than negtive group. At last,we analyzed the CTY apoptosis rate treated with Hoechst-33258and found that CTYhad a characteristic condensation of the chromatin as well as dissolution of thenucleolar structure and nuclear fragmentation, nuclear chromatin shrinkage aftertransfected with pCAGGS-VP1. In contrast, the control group cells had severalhyperchromatic nuclei, most nuclear staining shallow, and the nuclear size and shapewas homogeneous. The cells falled off the wall of the bottle after tansfected withpCAGGS-VP1and selected same size vision in two pictures to statistic the apoptosisrate found that the cells number transfected with pCAGGS is2-3times higher thanthe group that transfected with pCAGGS-VP1.4. The truncated mutation of FMDV VP1is constructed and subcloned intoexpression vector pCAGGS by restriction endonuclease EcoR I and Xho I, and therecombinant plasmid was confirmed by sequencing and restriction endonucleasedigestion.5.The truncated FMDV VP1, full lenghth VP1and pCAGGS was transfectedrespectively by Lipofectamine2000into CTY and detected by flow cytometry withAV-PI staining, the result found that the full-length VP1and VP1-2(181-360) couldinduce apoptosis but the empty vector pCAGGS and VP1-1(1-213), VP1-3(331-510),VP1-4(481-639) induced apoptosis was not obvious.[Conclusion] This paper revealed that FMDV VP1could induce apoptosis in CTY through different methods, and the apoptosis rate was2-3times highercompared with the control group. Further study found that the region of213-330aa ofVP1protein was important for its induction of apoptosis in CTY.