应用患者恢复期血清初步筛选猪链球菌体内诱导表达抗原

Primary screen of in vivo induced antigen of streptococcus suis with convalescent serum

目的利用患者恢复期血清对中国猪链球菌2型05ZY/H33强毒株体内诱导表达抗原进行初步筛选,并对建立的05ZY/H33菌株基因组表达文库及体内诱导表达抗原筛选平台的可行性进行初步验证。方法提取05ZY/H33菌基因组DNA,由Sau3AⅠ不完全酶切后回收0.5～3kbDNA片段,插入经其同尾酶BamHⅠ酶切及去磷酸化处理的pET-30a/b/c表达载体系统,转化大肠埃希菌BL21(DE3),构建05ZY/H33菌株基因组表达文库。将收集的患者恢复期血清等体积混合,经体外培养的05ZY/H33和BL21(DE3)全细胞及细菌超声裂解物吸附处理后,获得吸收血清。用此血清采用免疫印迹法筛选IPTG诱导的基因组表达文库,寻找05ZY/H33强毒株的体内诱导表达抗原。结果 05ZY/H33强毒株的基因组文库构建共得到3.2×104个重组子,重组阳性率达95%。随机挑选文库中的2000个重组子运用吸附处理后的患者恢复期血清对其进行体内诱导抗原的初步筛选,共筛到5个阳性克隆,经测序鉴定和生物信息学分析显示5个克隆子包含了05ZY/H33基因组的7个开放阅读框(ORF),其编码产物均参与细菌新陈代谢、复制增殖及损伤修复等重要的生命活动,极有可能为05ZY/H33强致病株的体内诱导抗原。结论本实验成功建立了运用中国猪链球菌2型05ZY/H33强毒株患者恢复期血清进行菌株体内诱导表达抗原筛选的技术平台,为05ZY/H33菌株体内诱导抗原的系统筛选奠定了基础。

The aim of the current article is to primarily screen the in vivo induced antigen of highly virulent Chinese streptococcus suis type 2 strain from pooled human adolescent serum,and to validate the feasibility of establishing genome expression library of strain 05ZY/H33 and in vivo induced antigen screen platform.Genomic DNA of 05ZY/H33 stain was extracted,with 0.5-3 kb DNA segments recycled after incomplete digestion with Sau3A I.The DNA segments were inserted into pET-30 a/b/c expression vehicle system which was digested by its isocaudarner BamHⅠand dephosphorylated,then introduced into E.coli BL21（DE3） to establish genome expression library of strain 05ZY/H33.The collected convalescent serum were pooled with equal volume of proteinase inhibitors,and then adsorbed by whole cell and ultrasound lysates from in vitro cultivated 05ZY/H33 and BL21（DE3） strain to produce adsorbed serum.The serum was applied to screen IPTG-induced genome expression library by using immunoblotting to explore in vivo induced antigen of 05ZY/H33 virulent strain.Total of 3.2×104 recombinants were acquired from established genome library of 05ZY/H33 virulent strain,with recombinant positive rate of 95%.We randomly selected 2 000 recombinants from the library to primarily screen in vivo induced antigen using convalescent serum,with 5 positive clones screened,and 7 open reading frame（ORF） were confirmed in these 5 clones after sequencing and bioinformatics analysis.The coding products of the 5 clones were involved in critical bacterial activities such as metabolism,replication,growth and repair,which was possible to be in vivo induced antigen of 05ZY/H33 virulent strain.In conclusion,the platform of in vivo induced antigen of streptococcus suis type 2 05ZY/H33 virulent strain is successfully established using convalescent serum,which could serve as the foundation of systemic screening of 05ZY/H33 strain in vivo induced antigen.