Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlue-Script II KS⁺ vector (pBSLTR), and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the lacZ gene; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACYCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 10^?4 to 10^?3, the frequency of spontaneously appearing blue colonies not exceeding 10^?9 to 10^?8. The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYCint) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the β-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them. gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).     

Evolution of errantiviruses of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>. Strategy 2: From retroviruses to retrotransposons

Drosophila melanogaster retrotransposons of the gypsy group are considered to be potential errantiviruses. Their infectivity is caused by the functional activity of the third open reading frame (ORF3) encoding the Env protein, which was probably captured from baculoviruses. Mobile genetic elements (MGEs) of the gypsy group can be conventionally divided into three subgroups: with three ORFs, with a defective ORF3, and without the ORF3. To establish the patterns of evolution of gypsy retrotransposons in D. melanogaster, the members of the three subgroups were examined. Structural analysis of retrotransposons opus and rover, which carry a defective ORF3, as well as retrotransposons Burdock, McClintock, qbert, and HMS-Beagle, which lack the ORF3, suggests that the evolution of these MGEs followed the pattern of loosing the ORF3. At the same time, an MGE of the same subgroup, Transpac, may be an ancestral form, which had acquired the env gene and gave rise to the first errantiviruses. The capture of the ORF3 by retrotransposons provided their conversion to a fundamentally new state. However, the ORF3 in the genome is not subjected to strong selective pressure, because it is not essential for intragenomic transpositions. Because of this, the process of its gradual loss seems quite natural.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.     

Insulator and Ovo proteins determine the frequency and specificity of insertion of the gypsy retrotransposon in Drosophila melanogaster

The gypsy retrovirus of Drosophila is quite unique among retroviruses in that it shows a strong preference for integration into specific sites in the genome. In particular, gypsy integrates with a frequency of >10% into the regulatory region of the ovo gene. We have used in vivo transgenic assays to dissect the role of Ovo proteins and the gypsy insulator during the process of gypsy site-specific integration. Here we show that DNA containing binding sites for the Ovo protein is required to promote site-specific gypsy integration into the regulatory region of the ovo gene. Using a synthetic sequence, we find that Ovo binding sites alone are also sufficient to promote gypsy site-specific integration into transgenes. These results indicate that Ovo proteins can determine the specificity of gypsy insertion. In addition, we find that interactions between a gypsy provirus and the gypsy preintegration complex may also participate in the process leading to the selection of gypsy integration sites. Finally, the results suggest that the relative orientation of two integrated gypsy sequences has an important role in the enhancer-blocking activity of the gypsy insulator.     

Characterization of the Envelope Glycoprotein of a Novel Filovirus,Lloviu Virus

Lloviu virus (LLOV), a novel filovirus detected in bats, is phylogenetically distinct from viruses in the genera Ebolavirus and Marburgvirus in the family Filoviridae. While filoviruses are known to cause severe hemorrhagic fever in humans and/or nonhuman primates, LLOV is biologically uncharacterized, since infectious LLOV has never been isolated. To examine the properties of LLOV, we characterized its envelope glycoprotein (GP), which likely plays a key role in viral tropism and pathogenicity. We first found that LLOV GP principally has the same primary structure as the other filovirus GPs. Similar to the other filoviruses, virus-like particles (VLPs) produced by transient expression of LLOV GP, matrix protein, and nucleoprotein in 293T cells had densely arrayed GP spikes on a filamentous particle. Mouse antiserum to LLOV VLP was barely cross-reactive to viruses of the other genera, indicating that LLOV is serologically distinct from the other known filoviruses. For functional study of LLOV GP, we utilized a vesicular stomatitis virus (VSV) pseudotype system and found that LLOV GP requires low endosomal pH and cathepsin L, and that human C-type lectins act as attachment factors for LLOV entry into cells. Interestingly, LLOV GP-pseudotyped VSV infected particular bat cell lines more efficiently than viruses bearing other filovirus GPs. These results suggest that LLOV GP mediates cellular entry in a manner similar to that of the other filoviruses while showing preferential tropism for some bat cells.     

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer

ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.     

白细胞介素6重组逆转录病毒载体的构建及鉴定

目的 构建特异性过表达大鼠IL-6基因的重组逆转录病毒载体,并在大鼠嗜铬细胞瘤PC12细胞和人胚肾HEK293细胞中检测IL-6的表达。方法以大鼠骨髓间充质干细胞mRNA为模板,经PCR获得目的基因IL-6,将其定向克隆到逆转录病毒载体pSEB-3H中,构建重组逆转录病毒质粒pSEB—IL-6,经脂质体分别转染到PC12细胞和HEK293细胞中,应用Real—timePCR和ELISA的方法在mRNA和蛋白质水平检测IL-6的表达变化。继而用HEK293细胞中包装获得的含有pSEB—IL-6的病毒颗粒进一步感染PC12细胞,Real—timePCR检测,IL-6mRNA的表达水平变化。结果PCR电泳及酶切鉴定证实目的基因正确克隆至逆转录病毒载体中,其基因序列与Genbank报道一致;Real—timePCR和ELISA结果均显示,逆转录病毒质粒pSEB—IL-6转染PC12细胞和HEK293细胞后,IL-6的表达水平较对照组显著上调;经pSEB—IL-6逆转录病毒颗粒感染的PC12细胞中,IL-6mRNA表达水平较对照组提高4倍。结论成功构建了特异性表达大鼠儿-6基因的重组逆转录病毒载体pSEB—IL-6,并获得了具有感染能力的逆转录病毒颗粒,感染真核细胞后可高表达IL-6,为进一步研究IL-6的功能及其在多种疾病中的免疫调节机制提供重要的分子手段。     

Development of Improved Adenosine Deaminase Retroviral Vectors

A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA⁻) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA⁻ severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.Retroviral vectors have been the most common gene transfer vehicles in clinical gene therapy trials (15). These vectors can integrate into the host genome to provide permanent transgene expression in the targeted cells (20). The first generation of retroviral vectors have been useful in demonstrating the feasibility of gene therapy approaches, but vectors capable of higher levels of gene transfer and transgene expression would be beneficial. For example, gene transfer levels achieved by first-generation retroviral vectors in large mammals (28) and in human gene therapy trials (7, 13) have been disappointing. There are at least two avenues for improving retroviral vectors. First, molecular changes can be made in the retroviral vector sequence. Second, different packaging cell lines could be tested to modify the host range, increase transduction in a given cell type, and/or render the virions resistant to inactivation by human complement.A clinically useful model for improving retroviral vector design is the vector LASN packaged in the amphotropic line PA317. PA317/LASN was the first therapeutic vector used in a gene therapy clinical trial (1). This vector has yielded gene transfer levels of generally less than 10% in peripheral blood T cells of adenosine deaminase-deficient (ADA⁻) severe combined immunodeficiency (SCID) patients. Two possibilities to improve this vector include eliminating the dominant selectable marker gene and changing the long terminal repeat (LTR) promoter to optimize expression. LASN, like many of the retroviral vectors used in clinical trials to date, contains two genes: the therapeutic gene (the ADA gene) and a dominant selectable marker gene (the bacterial neomycin phosphotransferase II gene; neo). Dominant selectable marker genes have historically been included to facilitate the generation, isolation, and titration of retroviral producer cell clones and to permit the evaluation and selection of successfully targeted cells. neo is the most commonly used selectable marker gene, although other genes have been used, including a mutant dihydrofolate reductase gene (dhfr) (19), the multidrug resistance gene (mdr) (10), and genes for cell surface markers such as cd24 (24) and the human nerve growth factor receptor (2). Vectors carrying dominant selectable marker genes, particularly those of nonhuman origin, have two theoretical disadvantages. First, careful analysis of some patients has revealed an immune response directed against the dominant selectable marker protein expressed from the retroviral integrant (20a, 25). Second, the more complex retroviral genomes required to express two separate genes may result in lower titers or suboptimal expression of the therapeutic gene product due to promoter interference (8, 29). On the other hand, cloning and determining the titers of useful retroviral vectors without selectable markers have been laborious. Using a recently developed rapid-screening procedure, we have been able to identify a number of “simple” ADA retroviral vectors which lack dominant selectable markers (23).Different packaging cell lines may also improve gene transfer of retroviral vectors into specific target cells. Retroviral vectors are limited by the host range specified by the envelope protein on the surface of the retrovirus. Most gene therapy trials have used retroviruses with a murine amphotropic (4070A) host range. However, packaging cell lines with the gibbon ape leukemia virus (GALV) envelope (PG13 cells) (18) and the cat endogenous virus RD114 envelope (FLYRD18 cells) (5) have become available; these may improve transduction frequencies into various target cell populations. For example, there is evidence that GALV-pseudotyped retroviral vectors may facilitate gene transfer into human peripheral blood T cells with greater efficiency than vectors with an amphotropic envelope (3). Packaging cell lines derived from murine cells have the additional disadvantage that they produce retroviruses which are inactivated by complement in human sera. Packaging cell lines of human origin (FLYA13 and FLYRD18) (5) produce vectors which are complement resistant. Testing both new simple retroviral vector designs and new packaging cells may therefore improve retrovirus-mediated gene transfer.We report the construction and characterization of three simplified ADA vectors by using either the Moloney murine leukemia virus (MLV) LTR, the myeloproliferative sarcoma virus (MPSV) LTR, or the SL3-3 LTR. We tested these vectors to determine which LTR provided the highest level of ADA expression in our target cells of interest: human ADA⁻ lymphohematopoietic cells. The ADA retroviral vector with the highest level of transduction/expression was then evaluated in different packaging cell lines including PG13, FLYA13, and FLYRD18.     

Molecular phylogeny and systematics of drosophila retrotransposons and retroviruses

Full classification of Drosophila melanogaster retrotransposons with long terminal repeats (LTR-retrotransposons) has been recomposed, and their evolutional analysis in sequenced genomes of different species of drosophila and other arthropods has been carried out. D. melanogaster LTR-retrotransposons are divided into three groups: gypsy (one, two, or three open reading frames (ORFs)), copia (one ORF), and BEL (one ORF). The gypsy group is divided into three subgroups. Subgroup I is underrepresented by retrotransposons-retroviruses with three ORFs and their derivatives, which have lost the env gene (ORF3). Subgroup II is underrepresented by retrotransposons with two ORFs, and subgroup III is underrepresented by retrotransposons with one ORF. A comparative analysis of homologs of gypsy group LTR-retrotransposons evidences that subgroups I and II are represented only in the genomes of Lepidoptera and Diptera. The gypsy group of LTR-retrotransposons with one and two ORFs is found in almost all genomes of arthropods. Most of the families of D. melanogaster gypsy group LTR-retrotransposons have close homologs in the genomes of other species of drosophila. A degree of identity of retrotransposons sequences is correlated with a degree of relation between species of drosophila, indicating vertical transmission of retrotransposons. Obvious cases of horizontal transfer of some mobile elements have been detected including retrotransposons without the env gene. Homologs of distinct ORFs of retrotransposons—genes gag and env—have been found. Gene-homolog of the gag gene—Grp (CG5680)—is under purifying selection, so it has an important function in drosophila genome.     

可表达随机12肽库的逆转录病毒表达系统的建立及评价

目的：建立可表达随机12肽库的逆转录病毒表达系统。方法：体外合成编码随机12肽的DNA片段;在最优化的实验参数和反应条件下将DNA片段克隆入带有EGFP标记的逆转录病毒载体后分批次电击转化大肠杆菌,合并转化所得菌液即为可表达随机12肽库的逆转录病毒原始载体库;半固体扩增法扩增该原始载体库,提取质粒并转染GP2-293包装细胞,在EGFP表达最强的时间点收集细胞培养上清,即为可表达随机12肽库的逆转录病毒库。结果：可表达随机12肽库的逆转录病毒原始载体库的库容量为3.14×10^6cfu;扩增后的逆转录病毒载体库滴度为5.2×109cfu/mL,库容量为2.34×1011cfu;转染了已扩增的载体库质粒后的GP2-293包装细胞可以成功地表达随机12肽库。结论：建立了可表达随机12肽库的逆转录病毒表达系统,为抗病毒寡肽的筛选以及进一步的深入研究奠定了良好的基础。     

共表达猪繁殖与呼吸综合征病毒GP5基因及猪γ-干扰素基因的腺病毒载体的构建与鉴定

利用PCR方法分别扩增猪繁殖与呼吸综合征病毒全长GP5基因（E蛋白）,EMCV的核糖体介入位点（IRES）序列及猪γ-干扰素（IFN-γ）基因全长序列,序列测定正确后用DNA重组法将三者串联后插入pAdenoVator-CM V5-IRES-GFP穿梭质粒中,形成的穿梭质粒plRES-GP5-IFN-γ用PmeⅠ线性化后,与腺病毒骨架载体pAdEasy-1共转化感受态大肠埃希氏菌BJ5183,经同源重组,构建成含有GP5基因和IFN-γ基因的重组腺病毒载体,pacⅠ酶切线性化充分暴露反向末端重复序列后,脂质体转染HEK293A细胞,借助GFP的表达可以在转染后的2～3天观察到包装病毒rAdeno-GP5-IFN-γ产生,7～10天出现病毒蚀斑。经PCR法及酶切证实各中间过程载体及最终的包装病毒中携带有目的基因,western-blot证实两基因在腺病毒中得到了表达。大肠杆菌内同源重组法能有效和较为方便的构建出含有目的基因的腺病毒载体rAdeno-GP5-IFN-γ,重组子能够在HEK293细胞中稳定扩增,病毒包装的成功为进一步研究PRRSVE蛋白的免疫效果及IFN-γ的作用奠定了基础。     

Mobilization of the gypsy and copia retrotransposons in Drosophila melanogaster induces reversion of the ovo dominant female-sterile mutations: molecular analysis of revertant alleles

The ovo locus is required for the maintenance of the female germ line in Drosophila melanogaster. In the absence of an ovo⁺ gene, males are completely normal but females have no germ-line stem cells. Three dominant mutations at the ovo locus, called ovo^D, were observed to revert towards recessive alleles at high frequency when ovo^D males were crossed to females of the strain y v f mal. We have found that this strain contains an inordinately high number of gypsy transposable elements, and crossing it with the ovo^D strains results in the mobilization of both gypsy and copia, with high-frequency insertions into the ovo locus: of 16 revertants examined 12 have gypsy and four have copia inserted at 4E, the ovo cytological site. Using gypsy DNA as a tag we have cloned 32 kb of wild-type DNA sequences surrounding a gypsy insertion and characterized molecular rearrangements in several independent revertants: in 10 of them gypsy appears to be inserted into the same site. The orientation of gypsy is strictly correlated with whether the neighbouring lozenge-like mutation appears in the revertants. A distal limit of the ovo locus was molecularly determined from the breakpoint of a deletion affecting closely flanking regions.     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

携带FMDV前导蛋白基因逆转录病毒载体的构建及其在牛肾细胞中的表达

以口蹄疫病毒株OA/58 RNA为模板, 反转录并扩增目的cDNA。通过分子克隆技术将前导蛋白编码序列Lab与逆转录病毒载体pBPSTR1连接, 将构建正确的重组载体命名为pBPSTR1-Lab。通过分别利用不同浓度的嘌呤霉素和四环素来确定最佳筛选浓度和最佳调控浓度, 结果显示嘌呤霉素的最佳筛选浓度为3 mg/mL, 四环素的最佳调控浓度为1 mg/mL。利用pBPSTR1-Lab和包装质粒pVSV-G双质粒瞬时转染Gp2-293包装细胞来获得重组逆转录病毒。利用重组逆转录病毒来感染牛肾细胞, 并连续筛选12天来获得阳性克隆。通过除去四环素来诱导目的基因在牛肾细胞中表达, 发现牛肾细胞病变死亡。经过PCR和蛋白质免疫印迹证实稳定表达前导蛋白的牛肾细胞系已经建立, 为今后研究前导蛋白致病机理提供了平台。     

携带FMDV前导蛋白基因逆转录病毒载体的构建及其在牛肾细胞中的表达

以口蹄疫病毒株OA/58 RNA为模板, 反转录并扩增目的cDNA。通过分子克隆技术将前导蛋白编码序列Lab与逆转录病毒载体pBPSTR1连接, 将构建正确的重组载体命名为pBPSTR1-Lab。通过分别利用不同浓度的嘌呤霉素和四环素来确定最佳筛选浓度和最佳调控浓度, 结果显示嘌呤霉素的最佳筛选浓度为3 mg/mL, 四环素的最佳调控浓度为1 mg/mL。利用pBPSTR1-Lab和包装质粒pVSV-G双质粒瞬时转染Gp2-293包装细胞来获得重组逆转录病毒。利用重组逆转录病毒来感染牛肾细胞, 并连续筛选12天来获得阳性克隆。通过除去四环素来诱导目的基因在牛肾细胞中表达, 发现牛肾细胞病变死亡。经过PCR和蛋白质免疫印迹证实稳定表达前导蛋白的牛肾细胞系已经建立, 为今后研究前导蛋白致病机理提供了平台。