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Wen-Jia Yang,Kang-Kang Xu,Wei Dou,Can Li,JinjunWang 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.3
3-Hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) is an important rate-limiting enzyme in the mevalonate pathway, which is critical for the synthesis of juvenile hormone and insect development. In this study, a full-length cDNA encoding HMGR (BdHMGR) was cloned from the oriental fruit fly, Bactrocera dorsalis (Hendel). The open reading frame of BdHMGR encoded 928 amino acids and shared a high degree of identity with other known HMGRs. The expression levels of BdHMGR were high during the early and lately pupal stages, and were low at the larval and mid pupal stages. In the adult stages, the highest level of BdHMGR was detected on day-10 of the adult lifecycle, and the expression levels in females were greater than those in males. The highest tissuespecific expression was in the ovary, followed by fat body and other tissues. Injection of double-stranded RNA (dsRNA) of BdHMGR into adult females significantly reduced BdHMGR transcript levels, and inhibited ovarian development and affected the expression of two yolk protein genes. After dissection, we found that one or two of the spheroids of ovaries from the BdHMGR dsRNA-injected flies were amorphous and reduced in size as compared with the controls. The results suggest that BdHMGR is required for ovarian development in B. dorsalis and may be a potential target for pest control.
( Xin Liu ),( Ying Dong ),( Jingquan Wang ),( Long Li ),( Zhenmin-zhong ),( Yun-pan Li ),( Shao-jun Chen ),( Yu-cai Fu ),( Wen-can Xu ),( Chi-ju Wei ) 한국미생물생명공학회(구 한국산업미생물학회) 2017 Journal of microbiology and biotechnology Vol.27 No.6
Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral, and artificial viral vectors. The objective of this study was to establish a potential approach for large-scale production of VSV-G. To this end, VSV-G was cloned with an N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Mut<sup>s</sup>) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, whereas the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by western blot analysis, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Furthermore, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G-mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with the pGL3-control DNA complex increased the luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with the pCMV-DsRed DNA complex improved the transfection efficiency into Ad293 by 10% and into HeLa cells by about 1-fold. In conclusion, these results demonstrate that VSV-G could be produced from P. pastoris with biofunctionalities, demonstrating that large-scale production of the viral glycoprotein is feasible.