cDNA cloning and prokaryotic expression of β-glucosidase in tea plant [Camellia sinensis (L.) O. Kutze]

2005 ◽  
Vol 2 (2) ◽  
pp. 107-111 ◽  
Author(s):  
Jiang Chang-Jun ◽  
Li Yuan-Hua ◽  
Fang Wan-Ping
Keyword(s):  
Fusion Protein ◽  
Camellia Sinensis ◽  
Prokaryotic Expression ◽  
Cdna Sequence ◽  
Expression System ◽  
Tea Plant ◽  
Aroma Precursors ◽  
Soluble Fusion Protein ◽  
Α Helix ◽  
Pleated Sheet

AbstractThe β-glucosidase gene has important effects on alcoholic aroma precursors and insect resistance of the tea plant [Camellia sinensis (L.) O. Kutze]. The complete cDNA sequence of β-glucosidase of the tea plant was cloned; its full length was 1475 bp, and shared 40–60% similarity with corresponding parts of the nucleotide sequence of β-glucosidase gene from other plants. Its secondary structure contains 14.33% α-helix, 25.43% β-pleated sheet and many functional amino acid domains. The β-glucosidase gene was cloned into the pET-32a expression system and expressed at high-efficiently in Escherichia coli BL21 (DE3); the molecular weight of expressed fusion protein was 63 kDa. The results of enzymic reaction showed that the fusion protein possessed normal bioactivity, and it could catalyse the dehydration of the glycosidic bond. The soluble fusion protein was expressed mainly in the cytoplasm.

Expression of Recombinant Human Cytomegalovirus Fusion Proteins pp150-pp65 Fragments and its Application

2013 ◽  
Vol 634-638 ◽  
pp. 1313-1318
Author(s):  
Yu Fen Jin ◽  
Yan Lei Li ◽  
Yan Hua ◽  
Xiao Gang Zhang ◽  
Ting Yu
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Human Cytomegalovirus ◽  
Colloidal Gold ◽  
Fusion Proteins ◽  
Prokaryotic Expression ◽  
Expression System ◽  
Serum Samples ◽  
Igg Antibody ◽  
Prokaryotic Expression System

Objective To evaluate the effectiveness of prokaryotic expression of fusion proteins pp150-pp65 of human cytomegalovirus (hCMV) for its application as antigen, the fusion protein of pp150-pp65 were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column for preparing the colloidal gold kit. Methods Using DNA from HCMV strain as template, the genes encoding pp150 and pp65 protein fragment were amplified by PCR technique, respectively. After confirmed by DNA sequence analysis, the recombinant plasmid pET28a-pp150-pp65 was transformed into E.Coil.BL21(DE3) and induced to express with IPTG. The expressed fusion protein was characterized by SDS-PAGE and western blot after purified, then we used the purified fusion protein to develop combined detection kit of IgM/IgG antibody against HCMV (colloidal gold method) with Beijing Innovita Bio-tech Co., Ltd for detecting the samples, compared with the imported kits (ELISA). Results The gene of fusion fragment pp150-pp65 was correctly amplified and the recombinant vector was successfully constructed. The purified protein with the molecular weight of 45KD had good antigenicity by western blot. The protein was subjected to assay with an ELI SA capture kit in its specific and sensitive assay based on colloidal gold nanoparticles, testing of 600 serum samples indicated that this kit had a sensitivity of 92.7%;, specificity of 83.1%, crude consistency o f 90.2%, compared to the imported HCMV-IgG kit; the sensitivity of the kit was 88.1%, specificity was 89.2%, coarse consistency was 88.5%, compared to the imported HCMV-IgM kit; Conclusion In this experiment, the HCMV antigen with high purity and specificity (pp150-pp65 recombinant protein) was prepared effectively through genetic engineering technology. Compared to imported reagents, the colloidal gold kit consisting of fusion protein in a capture assay had high sensitivity and specificity. Preliminary clinical use warrants further development and use of this kit. Furthermore, it provides a technological basis for detection of HCMV in different stages of clinical infection.

scholarly journals The recombinant anti-TNF-α fusion protein ameliorates rheumatoid arthritis by the protective role of autophagy

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Xiaole Chen ◽  
Kaimei Nie ◽  
Xin Zhang ◽  
Shuangyu Tan ◽  
Qingmei Zheng ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Fusion Protein ◽  
Prokaryotic Expression ◽  
High Efficiency ◽  
Expression System ◽  
Inflammatory Factors ◽  
Cytokine Signaling ◽  
Drug Study ◽  
Tnf Α ◽  
Prokaryotic Expression System

Abstract The currently used anti-cytokine therapeutic antibodies cannot selectively neutralize pathogenic cytokine signaling that cause collateral damage to protective signaling cascades carrying the potential for unwanted side effects. The variable domains of heavy-chain only antibodies (HCAbs) discovered in Camelidae are stable and display to be fully functional in antigen-binding against variable targets, which seem to be attractive candidates for the next-generation biologic drug study. The purpose of our study was to establish a simple prokaryotic expression system for large-scale expression, purification, and refolding of the recombinant anti-tumor necrosis factor α (TNF-α) fusion protein (FVH1-1) from inclusion bodies. Over 95% purity of the recombinant anti-TNF-α fusion proteins was obtained by just one purification step in our developed prokaryotic expression system, while the results of surface plasmon resonance (SPR) established the high-efficiency potent binding ability of FVH1-1 to human TNF-α. The counteraction of TNF-α cytotoxic effect experiment on the mouse fibroblast fibrosarcoma cell line (L929) confirmed that the expressed FVH1-1 were able to selectively and highly combine with human recombinant TNF-α (hTNF-α) in vitro. Western blot results showed that FVH1-1 can inhibit the activation of caspase-9 and PARP, which are the apoptotic signaling pathway proteins activated by hTNF-α. Meanwhile, lysosome autophagy signaling pathways stimulated by hTNF-α were inhibited by FVH1-1, which down-regulated the expression of LC3II/LC3I and up-regulated the expression of P62, indicating that the autophagy linked with TNF-α-induced apoptosis in response to rheumatoid arthritis. The results of the AIA rat model experiment presented that FVH1-1 can reduce the degree of joint swelling and inflammatory factors to a certain extent in vivo.

Protoplast Isolation and Fusion Induced by PEG with Leaves and Roots of Tea Plant (Camellia sinensis L. O. Kuntze)

2018 ◽  
Vol 44 (3) ◽  
pp. 463 ◽  
Author(s):  
Zhang PENG ◽  
Hua-Rong TONG ◽  
Guo-Lu LIANG ◽  
Yi-Qi SHI ◽  
Lian-Yu YUAN
Keyword(s):  
Camellia Sinensis ◽  
Protoplast Isolation ◽  
Tea Plant ◽  
Leaves And Roots

Cloning and Expression Analysis of Auxin Efflux Carrier GeneCsPIN3in Tea Plant(Camellia sinensis)

2016 ◽  
Vol 42 (1) ◽  
pp. 58 ◽  
Author(s):  
Bo WANG ◽  
Hong-Li CAO ◽  
Yu-Ting HUANG ◽  
Yu-Rong HU ◽  
Wen-Jun QIAN ◽  
...  
Keyword(s):  
Expression Analysis ◽  
Camellia Sinensis ◽  
Tea Plant ◽  
Cloning And Expression ◽  
Auxin Efflux Carrier

Low temperature effects on carotenoids biosynthesis in the leaves of green and albino tea plant (Camellia sinensis (L.) O. Kuntze)

2021 ◽  
Vol 285 ◽  
pp. 110164
Author(s):  
Ya-Zhuo Yang ◽  
Tong Li ◽  
Rui-Min Teng ◽  
Miao-Hua Han ◽  
Jing Zhuang
Keyword(s):  
Low Temperature ◽  
Camellia Sinensis ◽  
Temperature Effects ◽  
Tea Plant
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