褐黄血蜱Hf05基因的克隆及表达分析
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摘要
目的对褐黄血蜱Hf05基因进行克隆及表达分析。方法基于褐黄血蜱中肠转录组文库中的Contig16575序列设计引物,RACE法扩增Hf05 cDNA全长,利用生物信息学软件进行分析;利用qRT-PCR分析褐黄血蜱Hf05基因在不同发育阶段、不同吸血状态下的雌成蜱和不同组织的表达水平。结果 Hf05 cDNA全长1 058 bp,ORF长531 bp,编码176个氨基酸;其表达情况为:饱血雌成蜱>饱血若蜱>饱血幼蜱>雄成蜱(P<0.05),饱血雌成蜱>3/4饱血雌成蜱>1/2饱血雌成蜱>1/4饱血雌成蜱(P<0.05),饱血雌成蜱体壳>半饱血雌成蜱体壳>饱血雌成蜱唾液腺>半饱血雌成蜱唾液腺(P<0.05)。结论成功克隆褐黄血蜱Hf05基因,该基因编码176个氨基酸的Hf05蛋白,qRT-PCR检测Hf05基因在饱血褐黄血蜱雌成蜱高表达。
Objectives To clone and analyze the expression of the Hf05 gene in Haemaphysalis flava. Methods Primers were designed based on Contig16575 sequences from an H. flava midgut transcriptome library. Full-length cDNA of Hf05 was amplified using RACE and analyzed using bioinformatic software. The level of expression of Hf05 mRNA in H. flava in different life stages, feeding stages, and different tissues was quantified using qRT-PCR. Results Full-length cDNA of the Hf05 gene was 1058 bp in length, and the open reading frame(ORF) was 531 bp in length. The gene coded for 176 amino acid residues. The level of expression of Hf05 in H flava in different life stages, feeding stages, and different tissues was: engorged adult female ticks > engorged nymphs > engorged larvae > adult male ticks(P<0.05); fully engorged adult female ticks > 75% engorged adult female ticks > half engorged adult female ticks > 25% engorged adult female ticks(P<0.05); scuta of fully engorged adult female ticks > scuta of half engorged adult female ticks > salivary glands of fully engorged adult female ticks > salivary glands of half engorged adult female ticks(P<0.05). Conclusion The Hf05 gene was successfully cloned. The gene encodes 176 amino acid. Hf05 is a gene that is highly expressed in fully engorged adult female ticks.
Objectives To clone and analyze the expression of the Hf05 gene in Haemaphysalis flava. Methods Primers were designed based on Contig16575 sequences from an H. flava midgut transcriptome library. Full-length cDNA of Hf05 was amplified using RACE and analyzed using bioinformatic software. The level of expression of Hf05 mRNA in H. flava in different life stages, feeding stages, and different tissues was quantified using qRT-PCR. Results Full-length cDNA of the Hf05 gene was 1058 bp in length, and the open reading frame(ORF) was 531 bp in length. The gene coded for 176 amino acid residues. The level of expression of Hf05 in H flava in different life stages, feeding stages, and different tissues was: engorged adult female ticks > engorged nymphs > engorged larvae > adult male ticks(P<0.05); fully engorged adult female ticks > 75% engorged adult female ticks > half engorged adult female ticks > 25% engorged adult female ticks(P<0.05); scuta of fully engorged adult female ticks > scuta of half engorged adult female ticks > salivary glands of fully engorged adult female ticks > salivary glands of half engorged adult female ticks(P<0.05). Conclusion The Hf05 gene was successfully cloned. The gene encodes 176 amino acid. Hf05 is a gene that is highly expressed in fully engorged adult female ticks.
引文
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[10] Mckenna RV,Riding GA,Jarmey JM,et al.Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein[J].Parasite Immunol,1998,20:325-36.
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[12] Rodriguez M,Penichet ML,Mouris AE,et al.Control of Boophilus microplus population in grazing cattle vaccinated with a recombinant Bm86 antigen preparation[J].Vet paresitol,1995(57):339-49.
[13] Alzugaray MF,Parizi LF,Seixas L,et al.Molecular and functional characterization of Bm05br antigen from Rhipicephalus microplus[J].Ticks Tick Borne Dis,2017,2(8):320-9.
[14] Gao J,Luo J,Fan R,et al.Characterization of a concealed antigen Hq05 from the hard tick Haemaphysalis qinghaiensis and its effect as a vaccine against tick infestation in sheep[J].Vaccine,2009,27(3):483-90.
[15] Patton TG,Dietrich G,Brandt K,et al.Saliva,salivary gland,and hemolymph collection from ixodes scapularis ticks[J].J Vis Exp,2012,21(60):3894.
[16] Livak K,Schmittgen T.Analysis of relative geneexpression data using real-time quantitative-ePCR and the 2-△△CT Method[J].Methods,2001,25(4):402-8.
[17] Nilsson SC,Nita I,M?nsson L,et al.Analysis of binding sites on complement factor I that are required for its activity[J].J Biol Chem,2010,285(9):6235-45.
[18] Shahein YE,Molecular cloning and expression of a larval Hf05 from the cattle tick Boophilus annulatus[J].Vet Immun Immunopathol,2008,121(3-4):281-9.
[2] Cheng WY,Zhao GH,Jia YQ,et al.Characterization of Haemaphysalis flava (Acari:Ixodidae) from Qingling subspecies of giant panda (Ailuropoda melanoleuca qinlingensis) in Qinling Mountains (Central China) by morphology and molecular markers[J].PLoS One,2013,8(7):e69793.
[3] Ko S,Kang JG,Kim SY,et al.Prevalence of tick-borne encephalitis virus in ticks from southern Korea[J].J Vet Sci,2010,11(3):197-203.
[4] Moon S,Gwack J,Hwang KJ,et al.Autochthonous lyme borreliosis in humans and ticks in Korea[J].Osong Public Health Res Perspect,2013,4(1):52-6.
[5] Yu XJ,Liang MF,Zhang SY,et al.Fever with thrombocytopenia associated with a novel bunyavirus in China[J].N Engl J Med,2011,364(16):1523-32.
[6] Rar VA,Livanova NN,Panov VV,et al.Genetic diversity of Anaplasma and Ehrlichia in the Asian part of Russia[J].Ticks Tick Borne Dis,2010,1(1):57-65.
[7] 王卓,吴琼,杨慎江,等.长白山地区蜱类和鼠类中查菲埃立克体感染调查及16S rRNA序列分析[J].中国病原生物学杂志,2017,12(10):971-4,978.
[8] Noh Y,Lee YS,Kim HC,et al.Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea[J].Parasit Vect,2017,10(1):20.
[9] Yun SM,Lee YJ,Choi W,et al.Molecular detection of severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses in ixodid ticks collected from vegetation Republic of Korea[J].Ticks Tick Borne Dis,2016,7(5):970-8.
[10] Mckenna RV,Riding GA,Jarmey JM,et al.Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein[J].Parasite Immunol,1998,20:325-36.
[11] Willadsen P,Bird P,Cobon GS,et al.Commercialisation of a recombinant vaccine against Boophilus microplus[J].Parasitology,1995(110):43-50.
[12] Rodriguez M,Penichet ML,Mouris AE,et al.Control of Boophilus microplus population in grazing cattle vaccinated with a recombinant Bm86 antigen preparation[J].Vet paresitol,1995(57):339-49.
[13] Alzugaray MF,Parizi LF,Seixas L,et al.Molecular and functional characterization of Bm05br antigen from Rhipicephalus microplus[J].Ticks Tick Borne Dis,2017,2(8):320-9.
[14] Gao J,Luo J,Fan R,et al.Characterization of a concealed antigen Hq05 from the hard tick Haemaphysalis qinghaiensis and its effect as a vaccine against tick infestation in sheep[J].Vaccine,2009,27(3):483-90.
[15] Patton TG,Dietrich G,Brandt K,et al.Saliva,salivary gland,and hemolymph collection from ixodes scapularis ticks[J].J Vis Exp,2012,21(60):3894.
[16] Livak K,Schmittgen T.Analysis of relative geneexpression data using real-time quantitative-ePCR and the 2-△△CT Method[J].Methods,2001,25(4):402-8.
[17] Nilsson SC,Nita I,M?nsson L,et al.Analysis of binding sites on complement factor I that are required for its activity[J].J Biol Chem,2010,285(9):6235-45.
[18] Shahein YE,Molecular cloning and expression of a larval Hf05 from the cattle tick Boophilus annulatus[J].Vet Immun Immunopathol,2008,121(3-4):281-9.