Molecular Cloning and Bioinformatics Analysis of cyaA Gene of Vibrio alginolyticus Strain HY9901

[Objectives]This study was conducted to explore the biological functions of cyaA gene of Vibrio alginolyticus.[Methods]With DNA of V.alginolyticus HY 9901 as a template,primers were designed according to the sequence of cyaA gene,and the cyaA gene was amplified by PCR.Bioinformatics analysis was performed.[Results]The cyaA gene of V.alginolyticus HY9901 was 2529 bp in size,and encoded 842 amino acids.The molecular structure of CyaA protein was C_(4358)H_(6745)N_(1171)O_(1286)S_(35).Its theoretical molecular weight was 97.24167 kDa and the theoretical pI value was 5.56.It had no signal peptide and transmembrane domain.CyaA protein had three N-terminal glycosylation sites,one cAMP and cGMP-dependent protein kinase phosphorylation site,nine protein kinase C phosphorylation sites,nine casein kinase II phosphorylation sites,one tyrosine kinase phosphorylation site,seven N-terminal myristoylation sites,one pentenyl binding site and ten microbody C-terminal localization signal sites.Subcellular localization prediction showed that CyaA protein was mainly located in the nucleus and cytoplasm.Through multi-sequence alignment and phylogenetic tree construction,it was concluded that V.alginolyticus had high CyaA homology with other Vibrio species.cyaA of V.alginolyticus was clustered with Vibrio fluminensis and Vibrio marinisedimini,and they were closely related.The secondary structure of CyaA protein consisted ofα-helixes(43.11%),random coils(38.00%)and extended strands(14.49%).In protein network interaction,it was found that the proteins adjacent to CyaA protein were Crp-2,CpdA,Crr,PtsG-2,ANP67209.1,Crp-1,PykF,Pyk,RelA and Ndk.[Conclusions]This study provides a new idea for formulating strategies for the prevention and control of vibriosis.

绵羊肺源大肠杆菌cyaA基因缺失对小鼠肺泡巨噬细胞感染的影响

为研究cyaA基因编码的腺苷酸环化酶(AC)在绵羊肺源大肠杆菌XJ10感染小鼠肺泡巨噬细胞(MH-S)中的作用,本研究以绵羊肺源大肠杆菌XJ10野毒株(XJ10WT)为对照,通过荧光定量PCR检测XJ10 cyaA基因缺失株(XJ10ΔcyaA)在感染MH-S细胞过程中与黏附侵袭相关的motA、crl、fimH、pap基因的m RNA转录水平,结果显示,与XJ10WT相比,XJ10ΔcyaA在感染MH-S细胞过程中与黏附侵袭相关的motA、crl、fimH、pap基因的m RNA转录水平在感染后0和6 h时均极显著降低(P<0.001),crl和pap基因的m RNA转录水平分别在2 h和12 h时均极显著上调(P<0.001)。进一步通过吉姆萨染色观察XJ10WT和XJ10ΔcyaA黏附MH-S细胞情况,采用细胞和细菌共培养方式分析XJ10ΔcyaA对MH-S细胞的黏附及侵袭能力,并通过CCK-8法分析细菌对MH-S细胞增殖的抑制情况。结果显示,XJ10ΔcyaA和XJ10WT在感染后3 h黏附MH-S细胞的活菌数量达峰值并持续至5 h,随后逐渐下降,在感染后1 h~5 h的时间段内,XJ10ΔcyaA黏附MH-S细胞的活菌数量均极显著低于XJ10WT(P<0.001)。XJ10ΔcyaA和XJ10WT侵袭MH-S细胞的活菌数量随着感染时间的延长逐渐升高,5 h时达到峰值,后持续下降,XJ10ΔcyaA在感染后1 h~10 h侵袭MH-S细胞的活菌数量均显著或极显著低于XJ10WT。在感染MH-S细胞后的3 h~6 h,XJ10ΔcyaA对细胞增殖的抑制率均极显著低于XJ10WT(P<0.001)。上述结果首次表明,cyaA基因表达的AC可以显著上调绵羊肺源E.coli与黏附侵袭相关基因的转录水平,并参与绵羊肺源E.coli对MH-S细胞的黏附与侵袭,抑制MH-S细胞增殖。本研究为绵羊肺源E.coli致病机制的研究奠定了基础。

百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达

目的克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白。方法从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆入载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经8mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性。结果重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合。结论已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础。