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样例:牛疱疹病毒Ⅰ型ul49(VP22)基因的序列分析及其表达

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     以牛疱疹病毒I型 (BHV-I) 国内地方分离株感染的细胞培养物制备PCR模板,扩增出大小约0.77kb 的ul49基因完整编码区片段,将扩增片段克隆到pMD18-T载体中,获得含ul49基因的重组质粒pMD-VP22.采用双脱氧末端终止法进行序列测定,发现与国外Cooper株ul49基因序列完全一致,说明ul49基因相当保守.进一步将BHV-I ul49完整编码区片段插入原核表达载体pET-28a和真核表达载体pEGFP-C1中,获得分别与6xHis融合的原核表达质粒pET- 28aVP22和与EGFP融合的真核表达质粒pEGFPVP22.将pET-28aVP22转化大肠杆菌BL21(DE3),经IPTG诱导,SDS- PAGE电泳检测发现在38kDa处有一条特异性的表达带.利用脂质体介导,将pEGFPVP22转染PK-15细胞,经G418加压筛选,获得稳定表达 VP22的细胞克隆.在倒置荧光显微镜直接检测未固定的活细胞,发现pEGFPVP22转染细胞能发出很强的荧光并主要集中在细胞核中,而对照载体 pEGFP- C1转染细胞的荧光分布于胞浆.

错误译文

    PCR templates were prepared by using the cell cultures infected by bovine herpes virus I (BHV-I) domestic local isolates and employed to amplify the fragments of ul49 gene complete coding region of about 0.77kb long, which were then cloned to the pMD18-T vector, producing the recombinant plasmid pMD-VP22 containing ul49 gene. Sequence analysis was carried out with dideoxy termination, and it was found be similar to the sequence of Cooper ul49 gene, suggesting that ul49 gene is rather conserved. By further inserting the fragments of BHV-I ul49 complete coding region into prokaryotic expression vector pET-28a and eukaryotic expression vector pEGFP-C1, 6 xHis-fused prokaryotic expression plasmid pET-28aVP22 and EGFP-fused eukaryotic expression plasmid pEGFPVP22 were obtained respectively. After transformation of E. coli BL21 (DE3) with pET-28aVP22, induction of IPTG and SDS-PAGE electrophoresis assay, it was found that there was a specific expression band of 38 kDa long. Mediated with lipid, the pEGFPVP22 was transfected to PK-15 cells and screened under G418 pressure, producing cell clones that could express VP22 stably. Observing the unfixed living cells directly through inverted fluorescence microscope, it was found that pEGFPVP22 transfected cells can radiate very strong fluorescence that was mainly concentrated in the nucleus, while the fluorescence from the control vector, pEGFP-C1 transfected cells, was distributed in the cytoplasm.

修改后译文

     PCR templates were prepared by using the cell cultures infected by the domestic local isolates of bovine herpes virus I (BHV-I), and the complete coding region fragments of ul49 gene of about 0.77kb was amplified by the PCR templates..Then the amplified fragments were cloned to the pMD18-T carrier to obtain the recombinant plasmid pMD-VP22 containing ul49 gene. Sequence analysis was carried out by double deoxidation termination method, and the sequence was in full accord with the sequence of the ul49 gene of foreign Cooper strain, which suggested that ul49 gene was rather conserved. Furthermore, the complete coding region fragments BHV-I ul49 was inserted into prokaryotic expression vector pET-28a and eukaryotic expression vector pEGFP-C1, then 6 xHis-fused prokaryotic expression plasmid pET-28aVP22 and EGFP-fused eukaryotic expression plasmid pEGFPVP22 were obtained respectively., The plasmid pEGFPVP22 was transformed into escherichia coli BL21(DE3) and induced by IPTG, then it was found that there was a specific expression band of 38 kDa by SDS-PAGE electrophoresis assay. Mediated by liposome, PK-15 cells were transfected by pEGFPVP22 and the cell clones that could express VP22 stably were obtained after the pressure selection of G418. Observing the unfixed living cells directly through inverted fluorescence microscope, it was found that pEGFPVP22 transfected cells could radiate very strong fluorescence that was mainly concentrated in the nucleus, while the fluorescence of the control pEGFP-C1 transfected cells was distributed in the cytoplasm.


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