嗜对虾细胞的高效报告基因病毒表达系统研究及其在转基因对虾上的应用

Up to date, no adult transgenic penaeus shrimps have ever been successfully produced due to the lackage of effective gene transfer and expression technology in the fertilized eggs or early embryos of penaeous shrimps. To overcome this, the present project will be performed using green fluorescent protein (GFP/eGFP) as reporter gene and in vitro cultured shrimp primary cells as screening tool. This study aims to improve the gene transfer and expression efficiency of the commercial retrovirus (RV) and lentivirus (LV) expression systems mainly in the following ways, ① to introduce the envelope proteins of shrimp white spot syndrome virus (WSSV) into the packaged RV or LV GFP/eGFP virus in order to increase the tropism of the tested system to shrimp cells；② to introduce the SV40- or WSSV-derived DTS (DNA nuclear targeting sequence) into the RV or LV GFP/eGFP expression vectors in order to increase their nuclear import efficiency and thus the expression efficiency of the system tested；③ to introduce shrimp-derived highly active promoters into the RV or LV GFP/eGFP expression vectors in order to improve the expression efficiency of the tested system. Then, the reconstructed GFP/eGFP virus vector plasmids were transformed, or the pseudo-typed GFP/eGFP viruses were transduced, into the in vitro cultured shrimp primary cells and the corresponding expression or infection efficiencies were evaluated with inverted fluorescent microscope by detecting the GFP/eGFP signal. Then, based on the analysis of the above-mentioned data, penaeus shrimp cell-philic, highly efficient RV or LV gene transfer and expression systems were developed and then applied to live shrimps and try to develop adult transgenic penaeus shrimps. This study is the first try in the world, and the development of the above-mentioned shrimp-specific, highly efficient RV/LV reporter expression systems will lay a technical foundation in the breeding of disease-resistant and high productive transgenic penaeus shrimps as well as the functional analysis of shrimp genes in the future.

针对目前对虾受精卵或早期胚胎的基因转移与表达效率低，至今未成功获得成体转基因对虾的现状。本研究拟以绿色荧光蛋白为报告基因，以对虾原代培养细胞为工具，对现有泛嗜性反转录病毒（RV）和慢病毒（LV）表达系统进行改造，主要包括：①将对虾白斑病毒（WSSV）囊膜蛋白引入RV或LV包装病毒, 以提高系统的嗜对虾细胞性；②将SV40或WSSV病毒的DNA核靶向序列引入病毒表达载体，提高其从胞质转运入核的效率，以提高系统的表达效率；③将高活性对虾基因启动子引入病毒表达载体，以提高系统的表达效率。通过侵染或转染对虾细胞实验，并分析荧光的表达，建立嗜对虾细胞高效RV或LV报告基因转移与表达系统，并将其应用于对虾活体，以期获得成体转基因对虾。本研究在国内外是首次尝试，为今后培育高产抗病转基因对虾优良品种以及对虾基因功能研究奠定技术基础。

针对目前在对虾和对虾细胞中缺乏有效的基因转移与表达技术的现状，本项目分别以绿色(eGFP)和红色（mcherry）荧光蛋白为报告基因，以对虾原代培养细胞以及哺乳动物和昆虫细胞等为工具，成功对现有泛嗜性逆转录病毒和二代慢病毒基因转移与表达系统进行了改造，即①将对虾白斑综合征病毒（WSSV）囊膜蛋白(VP28和VP19)分别引入逆转录病毒和慢病毒的囊膜中, 以提高系统的嗜对虾细胞性；②将高活性对虾基因启动子（Ptctp）和对虾WSSV病毒启动子（Pie1-504）分别引入逆转录病毒和慢病毒的表达载体，以提高系统在对虾细胞的表达效率，最终成功建立了嗜对虾细胞的逆转录病毒和慢病毒报告基因转移与表达系统。发现，未改造的逆转录病毒表达系统不能侵染对虾细胞，观察不到绿色荧光。但是，改造过的逆转录病毒表达系统能够侵染对虾细胞，可观察到明显的弱的绿色荧光，但通过浸泡法或注射法感染糠虾幼体的实验未获成功；但是慢病毒明显优于逆转录病毒，即使是未改造的慢病毒也能感染对虾细胞，但是其感染效率明显低于改造过的慢病毒。已成功将改造过的慢病毒表达系统应用于对虾细胞和对虾活体中。在感染后的对虾体外培养血淋巴细胞中观察到强的荧光表达。在活体感染后对虾的血淋巴细胞和注射部位肌肉组织中观察到强的红色荧光表达。成功建立了嗜对虾细胞的昆虫杆状病毒GUS基因转移与表达系统，并成功应用于对虾外周血淋巴细胞的感染，观察到高GUS基因表达，并发现GUS报告基因比荧光蛋白更适用于对虾细胞。进一步优化了对虾细胞的培养基，完善了对虾外周血淋巴细胞和胚胎细胞的体外培养技术。成功克隆并分析了SV40病毒72 bp核靶向序列（DTS）和对虾WSSV病毒的4个候选DTS在对虾细胞中的DTS活性。建立了11R和TAT穿膜肽技术，成功介导eGFP蛋白穿膜进入对虾细胞中。以上研究成果为今后培育高产抗病转基因对虾优良品种以及对虾基因功能研究奠定了技术基础。