马链球菌兽疫亚种类M基因缺失株的构建及生物特性分析
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摘要
马链球菌兽疫亚种(Streptococcus equi ssp.zooepidemicus,SEZ)对集约化养猪业的发展危害严重,同时对养猪业的相关从业人员的健康具有潜在威胁。类M蛋白是该菌一种重要的表面蛋白和毒力因子,具有调理素功能,且能够抵抗的吞噬细胞对其的吞噬作用。本试验构建了类M蛋白基因(szp)的缺失株,在分子水平上对马链球菌兽疫亚种类M蛋白的粘附机制进行了研究,同时,通过动物模型评价了该突变株的毒力和免疫原性。因此本研究所构建的ATCC35246的类M蛋白基因缺失突变弱毒菌株,为该菌的致病机制及引起的猪链球菌病的防控进行了有益的探索。
1链球菌兽疫亚种ATCC35246株类M蛋白基因缺失株的构建
根据已发表的马链球菌兽疫亚种猪源ATCC35246株的类M蛋白基因序列,设计和合成一对基因步移引物,并以ATCC35246株的基因组DNA为模板,通过基因步移技术,扩增出类M蛋白左右两翼基因。为了敲除类M蛋白基因,以ATCC35246为始发菌株,构建基因重组质粒PUC-18,利用同源重组的原理构建了ATCC35246的类M蛋白基因缺失突变菌株(缺失了类M蛋白基因共1090bp)并对该菌株的致病力、小鼠模型的免疫保护作用进行了研究。结果表明,ATCC35246的类M蛋白基因缺失突变菌株对实验小鼠的致病性明显减弱;突变株能对小鼠提供良好的免疫保护,半定量RT-PCR显示,免疫鼠IL-2、IFN-γ基因的转录水平显著升高。所以经免疫的小鼠可以获得良好的被动免疫保护作用。为了进一步确定szp基因的具体作用,进行了马链球菌兽疫亚种szp基因缺失株的基因互补实验。实验证实基因互补株与野生株的生物特性基本一致。因此本研究所构建的ATCC35246的szp基因缺失突变弱毒菌株可作为预防ATCC35246感染的疫苗候选株,为最终研究制出马链球菌兽疫亚种链球菌的基因工程菌苗奠定基础。
2类M蛋白及其基因缺失株对HEp-2细胞的粘附作用
采用通用的模式细胞HEp-2细胞,研究马链球菌兽疫亚种ATCC35246株类M蛋白的粘附作用。结果显示,用重组的ATCC35246株类M蛋白预处理HEp-2细胞,当类M蛋白的量达到3.2μg/200μL,可显示为阳性;用重组表达的类M蛋白单抗处理类M蛋白,可抑制其对HEp-2细胞的粘附,在1.8μg/500μL能完全抑制它对HEp-2细胞的粘附,说
明其单抗对应的抗原表位在细胞粘附中具有决定性作用。同时发现,亲本株及szp基因缺失株均对HEp-2细胞有黏附和侵袭力,但szp基因缺失株黏附能力降低50%以上。电镜观察结果显示,黏附部位是细胞膜和细胞微绒毛,并观察链球菌内化现象。以上结果提示,上皮细胞是SEZ的定植细胞;菌株黏附后直接侵入细胞内是其穿过上皮细胞屏障的机制之一。
Streptococcus equi ssp.zooepidemicus made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry.M-like protein(szp) of Streptococcus equi ssp.zooepidemicus is a major surface protein that conveys the resistance to phagocytosis and it is a virulent factor with opsonsin function.In the present study,a szp-gene knock out mutant of Streptococcus equi ssp.zooepidemicus was constructed,at the molecular levels we investigated the adhensive machanism of M-like protein,evaluated the pathogenicity and innunogenicity of the mutant strain in animal model of infection.
To knock out the entire M-like protein gene of Streptococcus equi subsp zooepidemicus ATCC35246 strain via and construct a M-like protein-deleted mutant strain of Streptococcus equi ssp zooepidemicus.Two DNA segments,respectively derived from the upperstream 1178bp and 1172bp downstream of szp gene,were obtained by genome walking from gene DNA of of Streptococcus equi subsp zooepidemicus ATCC35246 strain, and these two segments were used to construct puc-18 deletion vector.The vector was then used to delete a 1090bp fragment of szp gene from a stain of SEZ(ATCC35246) through homologous recombination.The szp-knockout strain was evaluated for virulence and antigenicity in mice model of infection.Results showed,comparing with the wild type,the szp-knockout strain had a dramatically decreased LD50.Most mice innocuated with the szp-knockout strain can resis the challenge of the virulent strain.Semi-quantitative RT-PCR analysis showed a marked increased in the level of IL-2 and IFN-γmRNA in immunized mice with mutant strain.In the same time,a complementation assay was performed to detect the restoration of the wild-type phenotype from the szp gene knockout mutant.This mutant strain could be used as an attenuated vaccine candidate.
HEp-2 cells,a kind of typical cell model,was used in the adhesion and adhesive inhibition experiments to evaluate the adhesion function of M-like protein from Streptococcus equi ssp.zooepidemicus strain ATCC35246 by ELISA.The adhesion results showed that M-like protein could adhere to HEp-2 cells.Treating recombinant M-like protein with the monoclonal antibody can inhibit the adhesion of M-like protein to HEp-2 cells.When concentration of M-like protein was up to3.2μg/200μL,the complete adhesive was obtained.Pretreatment of M-like protein with the monoclonal antibody against purified recombinant M-like protein at 1.8μg/500μL could inhibit its adhesion completely; Adhesion counting assay demonstrated high adhesive levels of both strains.Scanning electron microscope observation further showed a dense adhering of ATCC35246 at cellular membrane and microvilli,Lysis counting assay revealed a invasion of ATCC35246 and weak invasion of ATCC35246△szp to HEp-2 cells.These findings suggested,firstly,the epithelial cell served as the colonizing site and an entrance in establishment of infection;secondly,the direct invasion into epithelial cells following their adhesion was one of their mechanisms of breaking through the epithelial cell barrier.
1链球菌兽疫亚种ATCC35246株类M蛋白基因缺失株的构建
根据已发表的马链球菌兽疫亚种猪源ATCC35246株的类M蛋白基因序列,设计和合成一对基因步移引物,并以ATCC35246株的基因组DNA为模板,通过基因步移技术,扩增出类M蛋白左右两翼基因。为了敲除类M蛋白基因,以ATCC35246为始发菌株,构建基因重组质粒PUC-18,利用同源重组的原理构建了ATCC35246的类M蛋白基因缺失突变菌株(缺失了类M蛋白基因共1090bp)并对该菌株的致病力、小鼠模型的免疫保护作用进行了研究。结果表明,ATCC35246的类M蛋白基因缺失突变菌株对实验小鼠的致病性明显减弱;突变株能对小鼠提供良好的免疫保护,半定量RT-PCR显示,免疫鼠IL-2、IFN-γ基因的转录水平显著升高。所以经免疫的小鼠可以获得良好的被动免疫保护作用。为了进一步确定szp基因的具体作用,进行了马链球菌兽疫亚种szp基因缺失株的基因互补实验。实验证实基因互补株与野生株的生物特性基本一致。因此本研究所构建的ATCC35246的szp基因缺失突变弱毒菌株可作为预防ATCC35246感染的疫苗候选株,为最终研究制出马链球菌兽疫亚种链球菌的基因工程菌苗奠定基础。
2类M蛋白及其基因缺失株对HEp-2细胞的粘附作用
采用通用的模式细胞HEp-2细胞,研究马链球菌兽疫亚种ATCC35246株类M蛋白的粘附作用。结果显示,用重组的ATCC35246株类M蛋白预处理HEp-2细胞,当类M蛋白的量达到3.2μg/200μL,可显示为阳性;用重组表达的类M蛋白单抗处理类M蛋白,可抑制其对HEp-2细胞的粘附,在1.8μg/500μL能完全抑制它对HEp-2细胞的粘附,说
明其单抗对应的抗原表位在细胞粘附中具有决定性作用。同时发现,亲本株及szp基因缺失株均对HEp-2细胞有黏附和侵袭力,但szp基因缺失株黏附能力降低50%以上。电镜观察结果显示,黏附部位是细胞膜和细胞微绒毛,并观察链球菌内化现象。以上结果提示,上皮细胞是SEZ的定植细胞;菌株黏附后直接侵入细胞内是其穿过上皮细胞屏障的机制之一。
Streptococcus equi ssp.zooepidemicus made substantial economic losses for pig industry and became a substantial threat to human health especially those involved in the pig industry.M-like protein(szp) of Streptococcus equi ssp.zooepidemicus is a major surface protein that conveys the resistance to phagocytosis and it is a virulent factor with opsonsin function.In the present study,a szp-gene knock out mutant of Streptococcus equi ssp.zooepidemicus was constructed,at the molecular levels we investigated the adhensive machanism of M-like protein,evaluated the pathogenicity and innunogenicity of the mutant strain in animal model of infection.
To knock out the entire M-like protein gene of Streptococcus equi subsp zooepidemicus ATCC35246 strain via and construct a M-like protein-deleted mutant strain of Streptococcus equi ssp zooepidemicus.Two DNA segments,respectively derived from the upperstream 1178bp and 1172bp downstream of szp gene,were obtained by genome walking from gene DNA of of Streptococcus equi subsp zooepidemicus ATCC35246 strain, and these two segments were used to construct puc-18 deletion vector.The vector was then used to delete a 1090bp fragment of szp gene from a stain of SEZ(ATCC35246) through homologous recombination.The szp-knockout strain was evaluated for virulence and antigenicity in mice model of infection.Results showed,comparing with the wild type,the szp-knockout strain had a dramatically decreased LD50.Most mice innocuated with the szp-knockout strain can resis the challenge of the virulent strain.Semi-quantitative RT-PCR analysis showed a marked increased in the level of IL-2 and IFN-γmRNA in immunized mice with mutant strain.In the same time,a complementation assay was performed to detect the restoration of the wild-type phenotype from the szp gene knockout mutant.This mutant strain could be used as an attenuated vaccine candidate.
HEp-2 cells,a kind of typical cell model,was used in the adhesion and adhesive inhibition experiments to evaluate the adhesion function of M-like protein from Streptococcus equi ssp.zooepidemicus strain ATCC35246 by ELISA.The adhesion results showed that M-like protein could adhere to HEp-2 cells.Treating recombinant M-like protein with the monoclonal antibody can inhibit the adhesion of M-like protein to HEp-2 cells.When concentration of M-like protein was up to3.2μg/200μL,the complete adhesive was obtained.Pretreatment of M-like protein with the monoclonal antibody against purified recombinant M-like protein at 1.8μg/500μL could inhibit its adhesion completely; Adhesion counting assay demonstrated high adhesive levels of both strains.Scanning electron microscope observation further showed a dense adhering of ATCC35246 at cellular membrane and microvilli,Lysis counting assay revealed a invasion of ATCC35246 and weak invasion of ATCC35246△szp to HEp-2 cells.These findings suggested,firstly,the epithelial cell served as the colonizing site and an entrance in establishment of infection;secondly,the direct invasion into epithelial cells following their adhesion was one of their mechanisms of breaking through the epithelial cell barrier.
引文
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