The Study Of Inhibition Of Porcine Endogenous Retrovirus Expression By RNA Interference And The Polyclonal Antibody Preparation

Part I Preparation of rabbit anti-porcine endogenous retrovirus P15E polyclonal antibodyBackground and ObjectionThe organs, tissues, and cells of swine have broad prospects been used as a donor source in clinical transplant. This has helped overcome the shortage of organs available for clinical transplant and provide a clinical treatment of some resistant nerve disorders just like the type-I insulin-dependent diabetes mellitus. Parkinson’s disease, Huntington’s disease, epilepsy and so on. The hepatocytes can also be used as a biotic component in the swine hepatocyte-based biartificial liver to treat the acute fulminant hepatitis liver function failure and a transitional measure to end-stage liver failure. Since1997, Dr. Clivepatienee (Bio Transplantxnc. Chief scientist) had reported that the porcine endogenous retrovirus (PERV) had infected the human cells cultured in vitro, it has attracted widespread attention from scholars and practitioners about the potential risk of PERV infection during swine xenotransplantation or hepatocyte-based biartificial liver.It was Patience who first confirmed that there are at least three subtypes of PERV that can infect the human cells in vitro. Takeuchi. Martin and some other scholars also prove that PERV can infect human cell lines in vitro.Since1998, Denner J had reported that one of the viral structural proteins, the transmembrane envelope protein (p15E), is the active component, and that purified p15E is able to inhibit a whole range of in yitro and in vivo immune responses. Besides, in the detection of PERV protein expression levels, no specific antibodies, that can detect the PERV P15E, can be seen in both domestic and international antibody market. Based on the PERV P15E antigen site reported abroad, this study designed and produced the polyclonal antibody of rabbit anti-pig PERV P15E, which has high sensibility and specificity in the detection of PERV protein expression levels. The research and development of this antibody will have prominent importance on detecting the expression and transmission of PERV in clinical organ transplantation and swine hepatocytc-based biartificial liver.MethodsWe got the epitopes of E1and E2protein sequences through literatures, based on which we traced back to the nucleic acid sequence of E1and E2, of P15E. The minimum length of P15E nucleic acid sequence is317bp. In order to enhance the antigencity of the nucleic acid sequence and the integrity of the epitope. we amplified100p nucleic acid of the anterior of P15E. and got the sequence of P15E coding region by PCR. After that, we connected it into the PEGX-4T-1vector, transforming it to the colon bacillus BL21(DE3).Through the self-induction method, the BL21(DE3) can express the fusion protein in recombinant plasmid, and then purified the protein through rubber tapping and recycling. Finally, we immunized the New Zealand rabbits with the fusion protein to prepare polyclonal antibodies and detected the activity and titer with Western blot and ELISΔ.ResultsThe rabbit-anti-pig polyclonal antibody can specifically detect the expression of P15E antigenic protein and P15E protein in PK15cells. However, the expression of P15E cannot be detected in HEK.293cells. which can prove the high specificity of the rabbit-anti-pig polyclonal antibody. Detection using ELISA revealed that the antibody titer drop degree is higher than1:100,000.ConclusionThe rabbit-anti-pig polyclonal antibody obtained in this study, possess high titer as well as specificity. It can become essential implement in detecting the expression and transmission of PERV in clinical organ transplant or swine hepatocyte-based biartificial liver. Part II Retrovirus-mediated RNA interference inhibition of PERV expression in Tibetan miniature pig fibroblastsBackground and ObjectionXenotransplants provide rich alternative sources of organs, tissues and cells that can be potentially used for the treatment of human diseases. Pig have become the first choice for xenotransplantation and nonhuman cell source provider for bioartificial liver because they are readily available and of high similarity in anatomy and physiology to human beings. However, the porcine endogenous retrovirus (PERV) was expressed in all of pig’s organs. tissues and cells. PERV is a gamma retrovirus that is integrated into porcine chromosomes. and large variations of its expression have been observed among the different pig breeds. The pig fibroblasts currently used are derived from the pig breeds which have a high background expression of PERV. Although PERV expression levels are significantly reduced after gene silencing via RNAi. these nuclear donor cells, which have a high expression of PERV, still present a highly potential risk for infection with human beings due to the "off-target effects" of RNAi technology.In order to obtain a pig fibroblasts with an extremely low PERV expression, preparing a safe nuclear donor cells for the next step to create transgenic pigs. We selected Chinese Tibetan miniature pig fibroblasts which have a low PERV mRNA expression. Meanwhile. applying the RNAi technology to obtain the pig fibroblasts that stably expressed the shRNA and the PERV at very low level. These modified pig fibroblasts may serve as safer nuclear donor cells for the generation of transgenic pigs.MethodsWe designed the small interfering RNAs (siRNAs) that target the highly conserved PERV-gag and PERV-pol domains. Then we constructed them into the expression vector pSUPERretro-GFP/Neo and transfected the PERV-HEK293cells. Quantitative real-time RT-PCR, Western blot and Immunocytochemistry are used to detect the change of PERV expression. We selected the most effective shRNA fragment and inserted it into the Tibetan miniature pig fibroblasts by retroviral expression vector for the resistance screening. At last, we applied the Quantitative real-time RT-PCR, Western blot and Immunocytochemistry to detect the PERV expression in positive clones Tibetan miniature pig fibroblasts. Date processing: Results were expressed as mean values士standard deviation(x±SD).A valtie of a=0.05was considered the standard of inspectability significant.and SPSS13.0software was used for statistics analysis.The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA.LSD method was used For multiple comparisons between groups for homogeneity of variance; otherwise Tamhane’s T2method was used.The independent samples T test was for two samples mean comparison.ResultsWe constructed the PERV-HEK293cell model expressing PERV gene,and this model can not only express PERV on the mRNA level,but also integrated in the genome.After transfecting the PERV-HEK293cells with shRNA. we utilized the SPSS13.0t0analyze the result of Quantitative real-time RT-PCR.One-way ANOVA showed that the transfection of shRNA can affect the PERV expression in PERV-HEK293cells (F=583.823,P<0.001). Groups multiple comparison(Tamhane’s, T2method)indicated that after the transfection of shRNA-pol3to PERV-HEK293cells,the relative PERV mRNA expression was (16.733±0.961)%.compared with negative control(91.467±6.312)%.indicating significant variation(P<0.001).The PERV protein expression in PERV-HEK293cells also decreased significantly.After inserted the shRNA-pol3into the Tibetan miniature pig fibroblasts by retrovirus vector,the results indicated high intcrference emciency of shRNA.Independent samples T test showed that there was a significant variation between the modified Tibetan miniature pig fibroblasts and wild fibroblasts (t=27.032,P<0.001).The PERV P15E protein expression in modified Tibetan miniature pig fibroblasts also decreased significantly.Conclusion The Tibetan miniature pig fibroblasts. stably expressed shRNA. was expressed lower PERV than before. They can serve as nuclear donor cells for the generation of safer transgenic pigs that stably express shRNA. Part III Knockdown of porcine endogenous retrovirus expression by multiple RNA interference in Chinese Experimental Miniature Pig fibroblastsBackground and ObjectionSwine tissues and cells contain porcine endogenous retrovirus (PERV). Multiple experiments, such as in vitro culture and infection-based examination. have confirmed that PERV can infect human cells. Van der Laan et al. transplanted swine islet cells into the enterocoelia of non-obese diabetic SCID mice and examined the level of PERV mRNA expression. They found that different types of tissues were infected with PERV, and theirs is considered the first report on cross-species infection and active internal replication of PERV following swine xenotransplantation.To minimize the possibility of the transmission of PERV scientists have used different strategies to inhibit or eliminate PERV expression. The best choice for inhibit the PERV mRNA and protein expression is to use RNA interference technology, as it mentioned in Part II. Many scientists using the virus vector to insert the siRNA into the pig fibroblas, making it expressed steadily in cells. For example, Dieckhoff etc. used the lentiviral vector to insert siRNA into the pig fibroblasts to inhibit the PERV expression. However, the viral vector was inserted into the cell the time we try to inhibit the PERV expression, which will bring about another kind of potential risk. The lentiviral vector is thought to be the most effective vector in transfection. widely used in genetic therapy. cell engineering and so on. However, as the uncertainty of the virus vector integration sites. it may result that the inserted gene’s mutation or even activation of the oncogene. Howe SJ etc. reported that the10SCID-â…¨ patients got the acute lymphoblastic leukemia. They were accepting the genetic therapy mediated by virus, and the letiviral vector caused the integration of the normal gene and the regulatory sequences in the upstream of LMO-2,35kb. Although many methods have been used to improve the safety of the letiviral vector by scholars, for example, altering the LTR sequences, inactivating the virus, separating the structural protein gene, packaging gene and so on. We can still not exclude the potential risk of tumorigenicity. Neyberg. S.L. etc. once reported that through the therapy of biartiticial liver, the cells for treatment can go into our bodies. The deformation movement of cells, filtration membrane damaged in the course of treatment can cause the immortal cells entering the human body. On the other hand, the gene segments released by the cells after splitting can go into the plasma of the patients. As we can see. viral vector bring about great potential risk in the modified gene cells.In order to inhibit the PERV expression stably and efficiently without the viral vector, we used the Perv-silencer expressing vector which has four promoters from different mammal species (hU6, hHl, mU6and h7SK). The different promoters were responsible for transcription of the corresponding shRNA structure unit, prevention of accidental recombinant, and effective transcription of every strand of shRNA. Thus, a single vector encoding four strands of shRNA and multiple interfering PERV-gag and PERV-pol domains was obtained. Besides, in the selection of pig fibroblasts, we chose the Chinese experimental miniature pig fibroblasts on the basis of our previous studies. The Chinese experimental miniature pig fibroblasts are with low PERV mRNA express and absence of the PERV-C provirus. We selected four siRNAs that target the PERV-gag and PERV-pol conserved domains:these siRNAs have the highest interference efficiency. The four siRNAs were integrated into a single vector and inserted into the selected fibroblasts by electroporation.The fibroblasts that were found to stably suppress PERV expression by resistance screening were used as a more secure nuclear donor cells for cultivate the transgenic pigs.MethodsIn this study, we designed8small interfering RNAs (siRNAs) that target the PERV-gag and PERV-pol conserved domains. We used them to transfect the PK15cells directly. Meanwhile, we used the Quantitative real-time RT-PCR and Western blot selecting four siRNAs which display the highest efficiency in inhibiting the expression of PERV. Then we constructed them into one single vector which was inserted into PK15cells via electroporation to validate the multiple RNA interference preliminarily. Quantitative real-time RT-PCR and immunofluorescence experiment were used to testify the variation of PERV expression. After previous screening and testing, we inserted the recombinant vector into the Chinese experimental miniature pig fibroblasts for multiple RNA interference. Quantitative real-time RT-PCR, Western blot and immunofluorescence were used to detect the PERV expression. We co-cultivated the HEK293cells with the supernate of the modified Chinese experimental miniature pig fibroblasts and detected the PERV expression of HEK293after co-cultivation by RT-PCR. Data processing:Results were expressed as mean values±standard deviation (x±SD), A value of a=0.05was considered the standard of inspectability significant. We used the SPSS13.0software for statistics analysis. The results of Quantitative real-time RT-PCR were analysed by one-way ANOVA. LSD method was used for multiple comparisons between groups for homogeneity of variance:otherwise Tamhane’s T2method was used.ResultsWe utilized the SPSS13.0to analyze the result of Quantitative real-time RT-PCR. One-way ANOVA showed that directly transfection of siRNA can affect the PERV expression in PK15cells (F-690.784, P<0.001). Groups multiple comparison(Tamhane’s, T2method) indicated that after transfection of siRNA-pol2, siRNA-po13, siRNA-gagl and siRNA-gag4to PK15cells, the relative PERV mRNA expression were (17.967±0.779)%,(17.917±1.094)%,(16.367±1.041)%,(18.100±0.832)%, compared with negative control (98.317±1.074)%, indicating significant variation (P<0.001). The PERV protein expression in PK15cells also decreased significantly. The PERV expression in PK15cells had been disturbed greatly after being transfected with the recombinant Perv-silencer expressing vector. Using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR demonstrated that transfecting PK15cells with recombinant Perv-silencer expressing vector affected the PERV expression (F=25615.576,P<0.001), Groups multiple comparison (LSD method) showed that after transfection with recombinant Perv-silencer expression vector to PK15cells, the relative PERV-gag and PERV-pol mRNA expression were (10.317±0.591)%and (9.733±0.568)%, compared with negative control (98.317±1.074)%. indicating significant variation (P<0.001).Immunofluorescence detection of its P15E protein expression was also significantly decreased; Finally, we stably transfected Chinese experimental miniature pig fibroblasts with recombinant Perv-silencer expression vector, using the one-way ANOVA to analyze the results of Quantitative real-time RT-PCR.The results showed that recombinant Perv-silencer expression vector can affect PERV expression in Chinese experimental miniature pig fibroblasts (F=5131.238, P<0.001), Groups multiple comparison (Tamhane’s. T2method) displayed that After Chinese experimental miniature pig fibroblasts were transfected with recombinant Perv-silencer expression vectors. the relative PERV-gagå'ŒPERV-pol mRNA expression were (7.100±1.037)%and (7.650±1.118)%. compared with negative control (96.112±2.623)%. indicating significant variation (P<0.001):Western blot and immunofluorescence detection of the P15E protein decreased significantly. No HEK293cells were infected with PERV when co-cultured with the supernate of the gene modified pig fibroblasts.ConclusionThe Chinese experimental miniature pig fibroblasts, obtained in this study, presented lower level of PERV expression compared with original cells and avoided the risk of PERV-A/C infection. Because no viral vectors were used in this experiment, so we avoided the potential risk of the viral vector going into the cells. Hence, they can serve as nucleus donor cells for transgenic pigs in subsequent studies.