本文關(guān)鍵詞：嗜水氣單胞菌外膜蛋白W基因的表達及其免疫原性分析，由筆耕文化傳播整理發(fā)布。

關(guān)鍵詞：基因拷貝數(shù),重組畢赤華根,脂肪酶

嗜水氣單胞菌外膜蛋白W基因的表達及其免疫原性分析
劉明智1,2  葉星1*  田園園1  馬冬梅1  張莉莉1  遲妍妍1,2  鄧國成1
(1. 中國水產(chǎn)科學研究院珠江水產(chǎn)研究所  廣東 廣州  510380)
(2. 上海海洋大學  上海  201306)

摘  要: 從患暴發(fā)性敗血病的草魚病灶處分離鑒定了嗜水氣單胞菌(Aeromonas hydrophila) Wp3菌株。以其基因組DNA為模板擴增外膜蛋白W基因(OmpW), 該基因全長為865 bp, 開放式閱讀框(ORF)為615 bp, 與標準株ATCC7966的OmpW基因的同源性為99.8%。根據(jù)ORF序列設計引物擴增OmpW成熟肽編碼序列并將其插入到表達載體pQE30中, 轉(zhuǎn)化大腸桿菌, 經(jīng)誘導可表達分子量為24.7 kD的帶His標簽的融合外膜蛋白His-W。用此融合蛋白免疫草魚, 所得草魚血清經(jīng)ELISA分析顯示呈現(xiàn)陽性反應, 說明重組蛋白能誘導產(chǎn)生抗體。采用實時熒光定量PCR分析草魚頭腎組織IgM基因表達水平的變化, 結(jié)果顯示免疫組IgM的表達量均明顯高于空白組, 其中低濃度免疫組(2 μg/g)與空白對照組的差異顯著(P<0.05), 說明融合蛋白可使草魚產(chǎn)生良好的免疫應答并上調(diào)抗體基因表達、產(chǎn)生高效抗體。保護性實驗顯示, 不同免疫劑量均可使免疫組獲得較高保護率(57%?86%)。結(jié)果顯示, 重組嗜水氣單胞菌外膜蛋白W可作為草魚嗜水氣單胞菌基因工程亞單位疫苗。

關(guān)鍵詞: 嗜水氣單胞菌, 外膜蛋白W, 克隆, 基因工程表達, 免疫原性, IgM, 保護率

生物谷推薦英文摘要：

Expression and immunogenicity analysis of the outer membrane protein W gene of Aeromonas hydrophila
LIU Ming-Zhi1,2  YE Xing1*  TIAN Yuan-Yuan1  MA Dong-Mei1  ZHANG Li-Li1 
CHI Yan-Yan1,2  DENG Guo-Cheng1

(1. Pearl River Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Guangzhou, Guangdong 510380, China)
(2. Shanghai Ocean University, Shanghai, 201306, China)

Abstract: The complete gene sequence of the outer membrane protein W (OmpW) was amplified from the genomic DNA of Aeromonas hydrophila Wp3 strain which was isolated from grass carp suffered hemorrhage. The OmpW gene was 865 bp in length, containing an open reading frame (ORF) of 615 bp. The cloned gene possessed high similarity with that of the standard strain ATCC7966 (99.8%). The se-quence encoding the mature peptide of OmpW was amplified and inserted into the expression vector pQE30. The recombinant vector was transformed into E. coli, and a 24.7 kD recombinant fusion protein His-W was expressed. ELISA analysis of the serum of grass carp immunized with His-W showed a positive immune reaction, suggesting production of the antibodies. Total RNA of the head kidney of the immunized grass carp was extracted and mRNA level of IgM was analyzed by qRT-PCR. Expression levels of IgM gene in the immunized group were higher than those of the control group, and significant difference (P<0.05) was found in the lowest protein concentration group (2 μg/g). These results showed that His-W was able to efficiently induce the immunized grass carp to produce antibodies. Protection experiments showed that the immunized groups had higher survival rates than those of the control groups (57%?86%). This study suggested that the recombinant protein His-W was a candidate vaccine for grass carp A. hydrophila disease.
Keywords: Aeromonas hydrophila, Outer membrane protein W, Cloning, Genetic engineering expression, Immunogenicity, IgM, Protection

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  本文關(guān)鍵詞：嗜水氣單胞菌外膜蛋白W基因的表達及其免疫原性分析，，由筆耕文化傳播整理發(fā)布。