鹦鹉热衣原体Mip基因原核表达载体的构建及多克隆抗体的制备
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摘要
目的克隆鹦鹉热衣原体巨噬细胞感染增强蛋白(Microphage infectivity potentiator,Mip)基因,构建原核重组质粒,诱导表达重组蛋白,并免疫BALB/c小鼠,制备多克隆抗体,为进一步研究其功能奠定了基础。方法提取鹦鹉热衣原体6BC基因组DNA,PCR扩增Mip基因,克隆至p ET-30a(+),构建原核表达载体p ET-30a(+)-Cps Mip。PCR、酶切及测序鉴定后,IPTG诱导p ET-30a(+)-Cps Mip在E.coli BL21中表达目的蛋白。融合蛋白纯化后免疫BALB/c小鼠,ELISA检测小鼠血清中抗Mip抗体的效价。结果 PCR扩增得到大小约为768 bp左右的Mip目的片段;经PCR、酶切及测序鉴定,证明构建的原核重组质粒中插入的片段为Cps Mip基因,测序结果经BLAST分析,与Genbank上登录序列完全一致。经IPTG诱导,重组工程菌表达一相对分子量(Mr)约为34 k D的目的蛋白;ELISA检测免疫小鼠血清效价为1:128 000。结论成功构建了鹦鹉热衣原体Mip基因原核表达载体,并表达了相应可溶性蛋白,制备了高效价的鼠抗Mip多克隆抗体。
Objective To construct a prokaryotic recombinant plasmid containing the gene encoding microphage infectivity potentiator( Mip) of Chlamydia psittaci( C. psittaci),and lay a basis for further study on the function of the recombinant protein.Methods The C. psittaci 6BC Mip gene was amplified by PCR and cloned into plasmid p ET-30( +),then transformed into E.coli BL21. After being identified by PCR,restriction enzymes cleavage and nucleotide sequencing,the positive strains were inducted by IPTG. Then the recombinant protein Mip was analyzed by SDS-PAGE and purified with Ni-NTA-His affinity chromatography.The purified recombinant Mip was used to immunize BALB/c mice,and the antibodies to Mip in mice sera were titrated with ELISA. Results A 768 bp fragment of C. psittaci Mip gene was obtained by PCR. The target gene was proved to be successfully inserted into p ET30( +) by PCR,restriction enzymes cleavage and nucleotide sequencing. The similarity was 100% between the inserted gene and Mip gene reported in Genbank by BLAST analysis. After the induction and purification,a recombinant protein about 34 KD was obtained. Specific humoral response was elicited in BALB/c mouse after immunization with the purified recombinant protein and the antibody titer in mice'sera was higher than 1: 128,000. Conclusions Prokaryotic expression plasmid p ET-30( +)-Cps Mip is successfully constructed,a molecular weight about 34 KDa protein is obtained and specific antibody is successfully prepared.
Objective To construct a prokaryotic recombinant plasmid containing the gene encoding microphage infectivity potentiator( Mip) of Chlamydia psittaci( C. psittaci),and lay a basis for further study on the function of the recombinant protein.Methods The C. psittaci 6BC Mip gene was amplified by PCR and cloned into plasmid p ET-30( +),then transformed into E.coli BL21. After being identified by PCR,restriction enzymes cleavage and nucleotide sequencing,the positive strains were inducted by IPTG. Then the recombinant protein Mip was analyzed by SDS-PAGE and purified with Ni-NTA-His affinity chromatography.The purified recombinant Mip was used to immunize BALB/c mice,and the antibodies to Mip in mice sera were titrated with ELISA. Results A 768 bp fragment of C. psittaci Mip gene was obtained by PCR. The target gene was proved to be successfully inserted into p ET30( +) by PCR,restriction enzymes cleavage and nucleotide sequencing. The similarity was 100% between the inserted gene and Mip gene reported in Genbank by BLAST analysis. After the induction and purification,a recombinant protein about 34 KD was obtained. Specific humoral response was elicited in BALB/c mouse after immunization with the purified recombinant protein and the antibody titer in mice'sera was higher than 1: 128,000. Conclusions Prokaryotic expression plasmid p ET-30( +)-Cps Mip is successfully constructed,a molecular weight about 34 KDa protein is obtained and specific antibody is successfully prepared.
引文
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[9]Bas S,Neff L,Vuillet M,et al.The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein,the macrophage infectivity potentiator,through TLR2/TLR1/TLR6 and CD14[J].J Immunol,2008,180(2):1158-1168.
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[12]Rockey DD,Chesebro BB,Heinzen RA,et al.A 28 k Da major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp.and Chlamydia trachomatis-cloning and characterization of the C.psittaci Mip-like gene[J].Microbiology,1996,142(4):945-953.
[13]Bas S,James RW,Gabay C.Serum lipoproteins attenuate macrophage activation and Toll-like receptor stimulation by bacterial lipoproteins[J].BMC Immunol,2010,138(1):11-25.
[14]Lundemose AG,Rouch DA,Penn CW,et al.The Chlamydia trachomatis Mip-like protein is a lipoprotein[J].J Bacteriol,1993,175(11):3669-3671.
[15]Lundemose AG,Kay JE,Pearce JH.Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection[J].Mol Microbiol,1993,7(5):777-783.
[2]Stenzel T,Pestka D,Choszcz D.The prevalence and genetic characterization of Chlamydia psittaci from domestic and feral pigeons in Poland and the correlation between infection rate and incidence of pigeon circovirus[J].Poult Sci,2014,93(12):3009-3016.
[3]Ling Y,Chen H,Chen X,et al.Epidemiology of Chlamydia psittaci Infection in racing pigeons and pigeon fanciers in Beijing,China[J].Zoonoses Public Health,2015,62(5):401-406.
[4]Fraeyman A,Boel A,van Vaerenbergh K,et al.Atypical pneumonia due to Chlamydophila psittaci:3 case reports and review of literature[J].Acta clinica Belgica,2010,65(3):192-196.
[5]Pannekoek Y,Visser C,Duim B,et al.Chlamydophila psittaci infections in the Netherlands[J].Drugs of today(Barcelona,Spain:1998),2009,45(Suppl B):151-157.
[6]Verminnen K,Vanrompay D.Chlamydophila psittaci infections in turkeys:overview of economic and zoonotic importance and vaccine development[J].Drugs Today(Barcelona,Spain:1998),2009,45(Suppl B):147-150.
[7]Knittler MR,Berndt A,Bocker S,et al.Chlamydia psittaci:new insights into genomic diversity,clinical pathology,host-pathogen interaction and anti-bacterial immunity[J].Int J Med Microbiol,2014,304(7):877-893.
[8]陆春雪,彭波,陈超群,等.抗沙眼衣原体MIP蛋白单克隆抗体的制备及特性鉴定[J].中国免疫学杂志,2014,30(1):80-84.
[9]Bas S,Neff L,Vuillet M,et al.The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein,the macrophage infectivity potentiator,through TLR2/TLR1/TLR6 and CD14[J].J Immunol,2008,180(2):1158-1168.
[10]Lu CX,Peng B,Wu YM,et al.Induction of protective immunity against Chlamydia muridarum intravaginal infection with the chlamydial immunodominant antigen macrophage infectivity potentiator[J].Microbes Infect,2013,(15):329-338.
[11]Zeng H,Hou S,Gong S,et al.Mapping immunodominant antigens and H-2-linked antibody responses in mice urogenitally infected with Chlamydia muridarum[J].Microbes Infect,2012,14(7-8):659-665.
[12]Rockey DD,Chesebro BB,Heinzen RA,et al.A 28 k Da major immunogen of Chlamydia psittaci shares identity with Mip proteins of Legionella spp.and Chlamydia trachomatis-cloning and characterization of the C.psittaci Mip-like gene[J].Microbiology,1996,142(4):945-953.
[13]Bas S,James RW,Gabay C.Serum lipoproteins attenuate macrophage activation and Toll-like receptor stimulation by bacterial lipoproteins[J].BMC Immunol,2010,138(1):11-25.
[14]Lundemose AG,Rouch DA,Penn CW,et al.The Chlamydia trachomatis Mip-like protein is a lipoprotein[J].J Bacteriol,1993,175(11):3669-3671.
[15]Lundemose AG,Kay JE,Pearce JH.Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection[J].Mol Microbiol,1993,7(5):777-783.