| Miniature pigs are considered to be the potential donors for xenotransplantation.However,porcine endogenous retrovirus(PERV)integrated in pigs genome,is likely to infect a wide spectrum of human and other mammalian cells in vitro.Although there is not final conclusion about the infection of PERV in vivo,PERV led to a panic among people.With the development of immunological rejection,PERV was regarded as the main hinder of xenotransplantation.Nowadays,Guangxi Bama(BM)which is used in medical research widely is the mature animal model,and is the potential donor for xenotransplantation with high expectations.Till now,the integration and effects of PERV-BM remains unknown.Therefore,we intend to study the integration characterization of PERV-BM in human genome and the effect of PERV on human cells.These studies will provide technical support for the development of BM.(1)Cloning and characterization of full-length PERV-BMBased on the sequence of PERV-BM(HM159246)and PERV-WZS(EF133960,GU980187),with the modified methods,two full-length PERV-BM were cloned,named PERV-BM2 and PERV-BM4.PERV-BM2 and PERV-BM4 with the intact gag,pol and env ORF produced 8860 bp and 8850 bp,respectively,displaying high homology to the PERV-A isolates from Wuzhishan miniature pigs(WZS)(GU980187,EF133960),BM(HM159246)and NIH(HQ536005).Based on the PCR products and phylogenetic tree,PERV-BM2 and PERV-BM4 belonged to subtype A,while the homology with PERV from domestic miniauture pigs ranged from 96.4% to 97.3%.To estimate the infection of PERV-BM for HEK293 cells,the diversity analyses were performed based on env gene and long terminal repeat(LTR)sequence.The 5'LTR of PERV-BM2/4 isolates comprised 2.5 copies of 39 bp repeats and two 18bp-21 bp sub-repeats followed by a 18 bp sub-repeat in U3 region,which conferred promoter activities in HEK293 cell line.Meanwhile,one 21bp-24bp-39 bp repeat sequence was found in 3'LTR,with the exception of1?3 bases identical to the former ones.Additionally,a 22 bp fragment was found in the upstream of the repeat sequences in 3'LTR,and the same insertion was also detected in the 3'LTR of GU980187.Besides,a curtailment of 12 nt(CCTTGTGGACAG)in the env gene of PERV-BM4 showed non-frameshift.Two bases of mutations and one base insertion were identified in the packaging signal of BM4.One base of mutation of BM4 in the env gene led to a premature stop codon in the N terminal of SU protein.All the molecular biology characteristics of PERV-BM2 and PERV-BM4 suggested that the infection,virion packaging and transcription level of PERV-BM might be lower than PERV-PK,especially PERV-BM4.This study is helpful for further study of PERV-BM infection and the construction of HEK293-PERV-BM.(2)Construction and identification of HEK293-PERV-BMTo study the infection,integration and effects on human cells of PERV-BM,HEK293-PERV-BM was constructed.With the activation of PHA or polybrene,PBMCs from 15 BMs were extracted and co-cultured with HEK293 cells,respectively.And PERV from only one BM(PERV-BM2)could infect HEK293 cells to establish HEK293-PERV-BM successfully,while the others were all failed to infect HEK293 cells.Meanwhile,HEK293-PERV-PK was established by the same procedure.Nest RT-PCR results showed that the transcriptional level of HEK293-PERV-BM was similar to each other and much lower than that of HEK293-PERV-PK with the same amount of RNA.Then during consecutive passages,the mutations occurred in PERV-BM displayed higher homology to PERV-PK and PERV-WZS than PERV-BM2.(3)Integration characterization of PERV from Bama miniature pigs in human genome With the modified LM-PCR and TAIL-PCR,among 3875 clones and 1483 clones,4 and 157 integration sites of PERV-BM and PERV-PK were identified from HEK293-PERV-BM and HEK293-PERV-PK,respectively,which displayed variations in position and formation.All of the PERV-BM integration sites were over ±5 kb from transcriptional start sites to Cp G islands,and showed the preference to integrating in human genome with the leader sequence as the junction sequences.And the genes which PERV-BM integrated in are also related with liver cancer,breast cancer,prostate cancer and Alzheimer's disease.PERV-PK showed the preference to integrating in human genome directly.The distance from the integration site of PERV-PK to the transcription start sites and Cp G islands was calculated,and 35.03% and 40.13% of the integration events occurred near to transcriptional start sites and Cp G islands,suggesting that PERV-PK tended to integrate in human genomic DNA directly around the transcriptional start sites and near Cp G islands of transcriptional active genes in the human chromosome.There is a common integration site between PERV-BM and PERV-PK,which is located in intron 3 of ROBO2 gene.(4)Effects of the integration of PERV on HERV-W expressions in HEK293 cellsTo find out whether the integration of PERV in HEK293 cells influence the expressions profile of HERV-W,based on the gene sequences of HERV-W in Gen Bank,the primers of HERV-W gag,pol,env,env from locus 7q21.2 genes(syncytin-1)as well as human ?-actin were synthesized,respectively,and were used to develop the means of SYBR Green I real-time reverse transcription-quantitative polymerase chain reaction(q PCR)to detect the m RNA expressions of these genes in HEK293 cells and PERV-infected HEK293 cells.The q PCR results showed that the m RNA expressions of HERV-W gag,pol,env and syncytin-1 changed after the integration of PERV-BM,and altered withdifferent passages.In the lower passages,the relative expressions of HERV-W gag,pol,env and syncytin-1 were a little higher or lower than HEK293,while in the higher passages,the relative expressions of HERV-W gag,pol,env and syncytin-1 increased by 3.72%?37.27%,and after passages 30 they decreased.The results of Western Blot showed that the protein expressions were similar to the m RNA expressions.(5)Effects of the integration of PERV on cell cycles and cell apoptosis as well as the expressions of some related genesThe cell cycles and cell apoptosis were altered after the integration of PERV-BM,and differed with different passages of HEK293-PERV-BM.To find out the mechanisms,q PCR was established to detect the m RNA expressions of cylin D1,CDK4,c-Myc,p53,p16 and k-Ras genes in different passages of HEK293-PERV-BM.Meanwhile,the protein expressions of cylin D1,CDK4,c-Myc and other genes was detected by Western blot.In lower passages of HEK293-PERV-BM,the cell cycle was arrested in S phase and G2/M phase as well as the cell apoptosis was inhibited.In higher passages of HEK293-PERV-BM,the cell cycle was arrested in S phase and the cell apoptosis was induced.The expressions of the related genes suggested that the cell cycle of lower passages might be regulated by Rb passway,while the cell cycle of higher passages might be regulated by Rb passway and p53 passway.These results provide the clues of potential infection and effects of PERV-BM.Our study focus on the molecular structure,infection,transcrption,integration and effects on HERV-W,cell cycle as well as cell apoptosis of PERV-BM.The results of this study provide theoretical and experimental basis for further evaluation of the security of PERV-BM in xenotranplantation and the development of BM industrialization. |
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