脊尾白虾组织蛋白酶L基因的克隆及其表达分析

根据本实验室构建的脊尾白虾(Exopalaemon carinicauda)血细胞全长cDNA文库获得的EST序列,利用RACE技术克隆获得脊尾白虾组织蛋白酶L基因的cDNA全长,命名为EcCatL基因.该序列全长1136 bp,包括5'非编码区24 bp,开放阅读框960 bp和3'非编码区152 bp,开放阅读框共编码319个氨基酸,预测相对分子量为35.30×103,理论等电点为5.27.同源性分析表明,脊尾白虾组织蛋白酶LEcCatL氨基酸序列与其它甲壳动物高度保守,与变色小长臂虾(Palaemonetes varians)及北极甜虾(Pandalus borealis) CatL的同源性分别为92％和76％.系统进化分析表明,EcCatL基因氨基酸序列与变色小长臂虾的CatL聚为一支.荧光定量PCR分析结果表明,EcCatL基因在血细胞、鳃、肝胰腺、肌肉、卵巢、肠、胃及眼柄中均有表达,其中肝胰腺中的相对表达量最高.感染鳗弧菌及WSSV后6h和12h,脊尾白虾血细胞和肝胰腺中EcCatL的表达量较对照组均极显著增加(P＜0.01),且具有明显的时间差异性,表明EcCatL基因在脊尾白虾免疫反应中具有重要作用.     

Cathepsin L Causes Proteolytic Cleavage of Chinese-Hamster-Ovary Cell Expressed Proteins During Processing and Storage: Identification,Characterization, and Mitigation

A stochastic approach of copurification of the protease Cathepsin L that results in product fragmentation during purification processing and storage is presented. Cathepsin L was identified using mass spectroscopy, characterization of proteolytic activity, and comparison with fragmentation patterns observed using recombinant Cathepsin L. Cathepsin L existed in Chinese hamster ovary cell culture fluids obtained from cell lines expressing different products and cleaved a variety of recombinant proteins including monoclonal antibodies, antibody fragments, bispecific antibodies, and fusion proteins. Therefore, characterization its chromatographic behavior is essential to ensure robust manufacturing and sufficient shelf life. The chromatographic behaviors of Cathepsin L using a variety of techniques including affinity, cation exchange, anion exchange, and mixed mode chromatography were systematically evaluated. Our data demonstrates that copurification of Cathepsin L on nonaffinity modalities is principally because of similar retention on the stationary phase and not through interactions with product. Lastly, Cathespin L exhibits a broad elution profile in cation exchange chromatography (CEX) likely because of its different forms. Affinity purification is free of fragmentation issue, making affinity capture the best mitigation of Cathepsin L. When affinity purification is not feasible, a high pH wash on CEX can effectively remove Cathepsin L but resulted in significant product loss, while anion exchange chromatography operated in flow-through mode does not efficiently remove Cathepsin L. Mixed mode chromatography, using Capto™ adhere in this example, provides robust clearance over wide process parameter range (pH 7.7 ± 0.3 and 100 ± 50 mM NaCl), making it an ideal technique to clear Cathepsin L. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2732, 2019     

Integrated Analysis of Transcriptomic,miRNA and Proteomic Changes of a Novel Hybrid Yellow Catfish Uncovers Key Roles for miRNAs in Heterosis

1. Download : Download high-res image (139KB)
2. Download : Download full-size image

Highlights

• •mRNA-seq, miRNA-seq, proteomes of P. fulvidraco, P. vachelli, hybrid Huangyou-1.
• •Predicted miRNA-mRNA-protein pairs were found and validated by qRT-PCR and PRM.
• •Immune, metabolism, digestion, absorption, proliferation, development generate heterosis.
• •High parental gene/protein with low parental miRNAs inherit from the mother or father.