Kozak序列引导的表达兔出血症病毒2型VP60蛋白重组腺病毒的构建及鉴定

doi:10.16431/j.cnki.1671-7236.2023.08.031

摘要/Abstract

摘要： 【目的】试验旨在以复制缺陷型人5型腺病毒(Ad5)为载体,构建Kozak序列引导的表达兔出血症病毒2型(RHDV2)VP60蛋白的重组腺病毒,为RHDV2新型疫苗研究提供依据。【方法】修饰、合成带有Kozak序列的VP60基因序列,将其与腺病毒穿梭质粒进行重组,转化大肠杆菌TOP10感受态细胞,构建重组腺病毒穿梭质粒,并利用酶切与测序对重组腺病毒穿梭质粒进行鉴定;采用Ad Max腺病毒包装系统,基于HEK293细胞进行穿梭质粒与腺病毒骨架质粒的定点同源重组,构建重组腺病毒Ad5-RHDV2-VP60并感染HEK293细胞,通过RT-PCR、Western blotting及间接免疫荧光试验(IFA)对重组腺病毒进行表达验证;利用Reed-Muench方法测定重组腺病毒的病毒滴度。【结果】重组腺病毒穿梭质粒酶切鉴定结果可见2条大小分别为3 895和1 752 bp的条带,测序比对结果显示,其与GenBank中公布的相应序列相似性＞99%,表明穿梭质粒构建成功。RT-PCR结果可见大小约为515 bp的特异性条带;Western blotting结果显示,大小约为60 ku的VP60蛋白特异性条带;IFA观察结果显示,感染重组腺病毒的HEK293细胞产生了绿色荧光,表明重组病毒能在HEK293细胞中表达VP60蛋白,且该蛋白具有良好的抗原性;病毒滴度测定结果显示,初代重组腺病毒的TCID₅₀为10^5.5/0.1 mL。【结论】本试验构建了含RHDV2 VP60基因的重组腺病毒,并成功表达了RHDV2 VP60蛋白,为进一步研究开发RHDV2疫苗提供了病毒模型。

关键词: 兔瘟; 兔出血症病毒2型(RHDV2); 重组腺病毒; HEK293细胞; Kozak序列

Abstract: 【Objective】 The purpose of this study was to construct a recombinant Adenovirus expressing Rabbit gemorrhagic disease virus type 2(RHDV2) VP60 protein leaded by Kozak sequence,based on a replication-defective Human adenovirus type 5(Ad5) and provide a basis for the research of RHDV2 new vaccine.【Method】 The VP60 gene with Kozak sequence were modified,synthesized and cloned to recombinant Adenovirus shuttle plasmid,and transferred into Escherichia coli TOP10 competent cells to construct Adenovirus shuttle vector,and the recombinants were analyzed and identified by restriction enzyme and sequencing.The recombinant Adenovirus Ad5-RHDV2-VP60 was packaged by the site-directed homologous recombination of shuttle plasmid and Adenovirus backbone plasmid,based on HEK293 cells using the Ad Max system.Then,the expression was verified by infecting HEK293 cells,and the recombinant Adenovirus was identified by RT-PCR,Western blotting and indirect immunofluorescence assay (IFA),and the titer of the recombinant Adenovirus was determined by Reed-Muench method.【Result】 Shuttle plasmid digestion result showed two bands with sizes of 3 895 and 1 752 bp,and the sequencing alignment results showed that it shared more than 99% similarity to the corresponding sequences published on GenBank,and the result showed that the shuttle plasmid was successfully constructed.The result of RT-PCR showed the 515 bp specific band,and the Western blotting result showed that the recombinant Adenovirus expressed a 60 ku VP60 protein.IFA results showed that HEK293 cells infected with the recombinant Adenovirus produced green fluorescence,and the VP60 specific protein could express in the HEK293 cells.The results of virus titer determination showed that the TCID₅₀of the primary recombinant Adenovirus was 10^5.5/0.1 mL.【Conclusion】 This study constructed a recombinant Adenovirus containing RHDV2 VP60 gene,which could successfully express RHDV2 VP60 with good antigenicity,and this study also provided a virus model for further research and development of RHDV2 vaccine.

Key words: rabbit hemorrhagic disease; Rabbit hemorrhagic disease virus type 2(RHDV2); recombinant Adenovirus; HEK293 cells; Kozak sequence

中图分类号:

S852.65

肖璐, 于吉锋, 罗燕, 谢晶, 叶勇刚, 吴学婧, 毛从剑, 李兴玉, 曹冶, 林毅, 潘梦, 叶健强, 魏勇, 康润敏. Kozak序列引导的表达兔出血症病毒2型VP60蛋白重组腺病毒的构建及鉴定[J]. 中国畜牧兽医, 2023, 50(8): 3336-3344.

XIAO Lu, YU Jifeng, LUO Yan, XIE Jing, YE Yonggang, WU Xuejing, MAO Congjian, LI Xingyu, CAO Ye, LIN Yi, PAN Meng, YE Jianqiang, WEI Yong, KANG Runmin. Construction and Identification of a Recombinant Adenovirus Expressing Rabbit Hemorrhagic Disease Virus Type 2 (RHDV2) VP60 Protein Leaded by Kozak Sequence[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3336-3344.

参考文献

[1] LE GALL-RECULE G,ZWINGELSTEIN F,BOUCHER S,et al.Detection of a new variant of Rabbit haemorrhagic disease virus in France[J].Veterinary Record,2011,168(5):137-138.
[2] 肖璐,于吉锋,林毅,等.中国首例兔出血症病毒2型(RHDV2/b/GI.2)的鉴定及病理学观察[J].中国畜牧兽医,2021,48(1):348-355. XIAO L,YU J F,LIN Y,et al.First detection of a type 2 Rabbit hemorrhagic disease virus (RHDV2/b/GI.2) in China and its pathological observation[J].China Animal Husbandry&Veterinary Medicine,2021,48(1):348-355.(in Chinese)
[3] ASIN J,REJMANEK D,CLIFFORD D L,et al.Circulation of Rabbit haemorrhagic disease virus type 2 in domestic and wild lagomorphs in Southern California,USA (2020-2021)[J].Transboundary and Emerging Diseases,2022,69(4):e394-e405.
[4] BYRNE A W,MARNELL F,BARRETT D,et al.Rabbit haemorrhagic disease virus 2(RHDV2;GI.2) in Ireland focusing on wild Irish hares (Lepus timidus hibernicus):An overview of the first outbreaks and contextual review[J].Pathogens,2022,11(3):288.
[5] BEN CHEHIDA F,LOPES A M,CôRTE-REAL J V,et al.Multiple introductions of Rabbit hemorrhagic disease virus Lagovirus europaeus/GI.2 in Africa[J].Biology,2021,10(9):883.
[6] CHEN M,FAN Z,HU B,et al.Pathogenicity of the newly emerged Lagovirus europaeus GI.2 strain in China in experimentally infected rabbits[J].Veterinary Microbiology,2022,265:109311.
[7] AGUAYO-ADÁN J A,ROUCO C,DELIBES-MATEOS M,et al.Lack of evidence for differences in the spread of classic (Lagovirus europaeus/GI.1) and novel (Lagovirus europaeus/GI.2) Rabbit haemorrhagic disease viruses in Europe and North Africa[J].Veterinary Research,2022,190(3):e1067.
[8] 曾红梅,潘雪晴,唐诗,等.兔出血症病毒2型VP60蛋白特异性单克隆抗体的制备[J].农业生物技术学报,2022,30(10):2053-2060. ZENG H M,PAN X Q,TANG S,et al.Preparation of monoclonal antibody specific to VP60 protein of Rabbit hemorrhagic virus type 2[J]. Journal of Agricultural Biotechnology,2022,30(10):2053-2060.(in Chinese)
[9] MOHAMED F,GIDLEWSKI T,BERNINGER M L,et al.Comparative susceptibility of Eastern cottontails and New Zealand White rabbits to classical Rabbit haemorrhagic disease virus (RHDV) and RHDV2[J].Transboundary and Emerging Diseases, 2022,69(4):e968-e978.
[10] KOZAK M.An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[J].Nucleic Acids Research,1987,15(20):8125-8148.
[11] GRZEGORSKI S J,CHIARI E F,ROBBINS A,et al.Natural variability of Kozak sequences correlates with function in a zebrafish model[J].PLoS One,2014,9(9):e108475.
[12] ARANGO D,STURGILL D,YANG R,et al.Direct epitranscriptomic regulation of mammalian translation initiation through N4-acetylcytidine[J].Molecular and Cellular Biology,2022,82(15):2797-2814.
[13] PETTY A P,DICK C L,LINDSEY J S.Translation of an atypical human cDNA requires fidelity of apurinepyrimidine repeat region and recoding[J].Gene,2008,414(1/2):49-59.
[14] 苏艳,王世民,邵俊高,等.Kozak序列对金黄色葡萄球菌黏附因子FnBPA-ADNA疫苗诱导小鼠免疫应答的影响[J].生物工程学报,2013,29(4):458-465. SU Y,WANG S M,SHAO J G,et al.Effect of Kozak sequence on mice DNA vaccine immunization of Staphylococcus aureus adhesion fibronectin-binding protein Fn BPA-A[J].Chinense Journal of Biotechnology,2013,29(4):458-465.(in Chinese)
[15] AMBROSINI C,DESTEFANIS E,KHEIR E,et al.Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency[J].Nucleic Acids Research,2022,50(18):10756-10771.
[16] DUBEY P.Gene therapy using Adenovirus vector[J].Journal of Molecular Biomarkers&Diagnosis,2021,12(7):480.
[17] WATANABE M,NISHIKAWAJI Y,KAWAKAMI H,et al.Adenovirus biology,recombinant Adenovirus,and Adenovirus usage in gene therapy[J].Viruses,2021,13(12):2502.
[18] 宋艳华,胡波,范志宇,等.兔出血症病毒2型SC株VP60基因工程疫苗研制及其与传统兔出血症病毒疫苗的交叉保护作用[J].江苏农业科学,2022,50(16):50-54. SONG Y H,HU B,FAN Z W,et al.Development of the VP60 gene engineering vaccine of Rabbit hemorrhagic hemorrhagic virus type 2 SC strain and its cross-protection with the traditional Rabbit hemorrhagic hemorrhagic virus vaccine[J].Jiangsu Journal of Agricultural Sciences,2022,50(16):50-54.(in Chinese)
[19] BOSCO-LAUTH A M,COMINSKY B,PORTER S,et al.A novel vaccine candidate against Rabbit hemorrhagic disease virus 2(RHDV2) confers protection in domestic rabbits[J].American Journal of Veterinary Research,2022,83(12):ajvr.22.05.0095.
[20] 李成琰.兔出血症病毒1型、2型二价组织灭活疫苗的制备及免疫效果评价[D].成都:西南民族大学,2022. LI C Y.Preparation of RHDV1 and RHDV2 bivalent tissue inactivated vaccine and its immunity evaluation[D].Chengdu:Southwest Minzu University,2022.(in Chinese)
[21] HRYCAK C P,WINDMANN S,BAYER W.Comparative evaluation of the vaccine efficacies of three Adenovirus-based vector types in the Friend retrovirus infection model[J].Journal of Virology,2019,93(21):e01155-19.
[22] 张倩,胡倩倩,王富珍,等.腺病毒载体疫苗研发进展[J].中国疫苗和免疫,2020,26(4):484-492. ZHANG Q,HU Q Q,WANG F Z,et al.Research and development of Adenovirus vectored vaccines[J].Chinese Journal of Vaccines and Immunization,2020,26(4):484-492.(in Chinese)
[23] 崔晓峰,孙志华,刘俊,等.腺病毒载体疫苗研究进展[J].中国病毒病杂志,2022,12(6):468-473. CUI X F,SUN Z H,LIU J,et al.Research progress on Adenovirus vector vaccines[J]. Chinese Journal of Viral Diseases,2022,12(6):468-473.(in Chinese)
[24] 柳云帆,吴小兵,阮力.腺病毒载体在疫苗研究中的应用[J].生物技术通讯,2011,22(4):552-558. LIU Y F,WU X B,RUAN L.Application of Adenovirus as vaccine vectors[J].Letters in Biotechnology,2011,22(4):552-558.(in Chinese)
[25] 董德金,王心仪,宗红,等.在无天然游离质粒产甘油假丝酵母中非整合表达合成咖啡酸[J].微生物学通报,2022,49(10):4104-4117. DONG D J,WANG X Y,ZONG H,et al.Synthesis of caffeic acid by episomal expression in Candida glycerinogenes without natural episomal plasmids[J].Microbiology China,2022,49(10):4104-4117.(in Chinese)
[26] XU H,WANG P,YOU J,et al.Screening of Kozak-motiflocated SNPs and analysis of their association with human diseases[J].Biochemical and Biophysical Research Communications,2010,392(1):89-94.
[27] 李巧彦,刘宗智,张婷,等.Kozak序列对nNOS基因表达及神经细胞分化的影响[J].石河子大学学报:自然科学版,2017,35(2):157-162. LI Q Y,LIU Z Z,ZHANG T,et al.The effect of Kozak sequence on the expression of nNOS and neuronal cell differentiation[J].Journal of Shihezi University(Natural Science),2017,35(2):157-162.(in Chinese)
[28] KAZEMI S A,NØRGAARD P,PALMQVIST E A,et al.Modulating heterologous protein production in yeast:The applicability of truncated auxotrophic markers[J].Applied Microbiology and Biotechnology,2013,97(9):3939-3948.