巴什拜羊、盘羊杂交羊重组SP-A蛋白对体外培养MO增殖的影响
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摘要
为研究巴什拜羊和盘羊杂交羊的重组肺表面活性物质相关蛋白A (rSP-A)对体外培养的绵羊肺炎支原体(MO)增殖的影响,本研究利用不同浓度(10μg/mL、20μg/mL和40μg/mL)巴什拜羊和盘羊杂交羊的rSP-A添加于MO培养液中进行体外培养,采用平板菌落计数和荧光定量PCR方法检测其对MO增殖的影响。平板菌落计数结果显示,巴什拜羊的3个rSP-A浓度组菌落数分别比对照组减少了8.35%(p>0.05)、23.04%(p<0.05)和40.03%(p<0.01);盘羊杂交羊的3个rSP-A浓度组菌落数分别比对照组减少了6.73%(p>0.05)、21.74%(p<0.05)和37.11%(p<0.01)。荧光定量PCR检测结果显示,添加rSP-A培养4 h后MO 16S rRNA基因拷贝数降至最低,巴什拜羊3个rSP-A浓度组16S rRNA基因拷贝数比对照组下降了72.15%(p<0.05)、78.81%(p<0.01)、81.48%(p<0.01);盘羊杂交羊的3个rSP-A浓度组的MO 16S rRNA基因拷贝数比对照组下降了26.26%(p>0.05)、76.62%(p<0.01)、80.83%(p<0.01)。研究表明,巴什拜羊和盘羊杂交羊的rSP-A对体外培养的MO具有明显的抑制作用。本研究比较了不同品种羊SP-A蛋白在抗MO感染中的作用,为进一步研究盘羊杂交羊易感MO的分子作用机制奠定基础。
To study the effects of recombinant pulmonary surfactant-associated protein A(rSP-A) from Bashiby sheep and Argal hybrid sheep on the replication of Mycoplasma ovipneumoniae(MO) in vitro, we cultured MO in the medium containing rSP-A at different concentrations(10 μg/mL, 20 μg/mL, and 40 μg/mL, respectively) and detected proliferation level of MO by the colony plate count and real-time PCR. The colony counts results showed that compared with the control group, the three experimental groups treated by Bashiby sheep rSP-A decreased by 8.35%(p>0.05), 23.04%(p<0.05), and 40.03%(p<0.01),respectively; in contrast, the Argal hybrid sheep rSP-A treatment groups decreased by 6.73%(p>0.05), 21.74%(p<0.05), and37.11%(p<0.01), respectively. The copy number of MO 16 S rRNA in three experimental groups reached the lowest at 4 hours after treatment, as shown by real-time PCR. The copy numbers of the three experimental groups treated by Bashiby sheep rSP-A decreased by 72.15%(p<0.05), 78.81%(p<0.01), 81.48%(p<0.01) compare with the control group, respectively; and that of Argal hybrid sheep rSP-A treatment groups 26.26%(p>0.05), 76.62%(p<0.01)、 80.83%(p<0.01) compare with the control group, respectively. These data demonstrated Bashiby sheep and Argal hybrid sheep rSP-A had marked inhibitory effect on the replication of MO in vitro. In this study, the effects of SP-A proteins from different breeds of sheep on anti-MO infection were compared, which laid a foundation for further study on the molecular mechanism of MO susceptibility.
To study the effects of recombinant pulmonary surfactant-associated protein A(rSP-A) from Bashiby sheep and Argal hybrid sheep on the replication of Mycoplasma ovipneumoniae(MO) in vitro, we cultured MO in the medium containing rSP-A at different concentrations(10 μg/mL, 20 μg/mL, and 40 μg/mL, respectively) and detected proliferation level of MO by the colony plate count and real-time PCR. The colony counts results showed that compared with the control group, the three experimental groups treated by Bashiby sheep rSP-A decreased by 8.35%(p>0.05), 23.04%(p<0.05), and 40.03%(p<0.01),respectively; in contrast, the Argal hybrid sheep rSP-A treatment groups decreased by 6.73%(p>0.05), 21.74%(p<0.05), and37.11%(p<0.01), respectively. The copy number of MO 16 S rRNA in three experimental groups reached the lowest at 4 hours after treatment, as shown by real-time PCR. The copy numbers of the three experimental groups treated by Bashiby sheep rSP-A decreased by 72.15%(p<0.05), 78.81%(p<0.01), 81.48%(p<0.01) compare with the control group, respectively; and that of Argal hybrid sheep rSP-A treatment groups 26.26%(p>0.05), 76.62%(p<0.01)、 80.83%(p<0.01) compare with the control group, respectively. These data demonstrated Bashiby sheep and Argal hybrid sheep rSP-A had marked inhibitory effect on the replication of MO in vitro. In this study, the effects of SP-A proteins from different breeds of sheep on anti-MO infection were compared, which laid a foundation for further study on the molecular mechanism of MO susceptibility.
引文
[1]晋大鹏,胡志帅,陈书明.肺表面活性物质相关蛋白A与肺部免疫防御研究进展[J].生物学杂志,2009,26(2):54-56.
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[4]Sano H,Kuroki Y.The lung collectins,SP-A and SP-D,modulate pulmonary innate immunity[J].Mol Immunol,2005,42(3):279-228.
[5]杜智慧,沈文,刘海燕,等.巴什拜羊与其杂交羊对绵羊肺炎支原体感染的比较[J].动物医学进展,2017,38(1):69-73.
[6]王继雪,孙延鸣.盘羊杂交羊SP-A基因克隆与真核表达[J].石河子大学学报(自然科学版),2018,36(3):309-313.
[7]张晓丽,高宝德,刘海燕,等.巴什拜羊重组SPLUNC1蛋白对体外培养的绵羊肺炎支原体生长的影响[J].中国预防兽医学报,2016,38(05):361-365.
[8]冷青文,李志远,鲁海富,等.盘羊体内绵羊肺炎支原体的分离和鉴定[J].中国预防兽医学报,2014,36(3):197-200.
[2]Piboonpocanun S,Chiba H,Mitsuzawa H,et al.Surfactant protein A binds Mycoplasma pneumoniae with high affinity and attenuates its growth by recognition of disaturated phosphatidylglycerols[J].J Biol Chem,2005,289(1):9-17.
[3]Zhang Shi-ping,Chen Yi,Potvin E,et al.Comparative signature-tagged mutagenesis identifies pseudomonas factors conferring resistance to the pulmonary collectin SP-A[J].PLoSPathog,2005,1(3):259-268.
[4]Sano H,Kuroki Y.The lung collectins,SP-A and SP-D,modulate pulmonary innate immunity[J].Mol Immunol,2005,42(3):279-228.
[5]杜智慧,沈文,刘海燕,等.巴什拜羊与其杂交羊对绵羊肺炎支原体感染的比较[J].动物医学进展,2017,38(1):69-73.
[6]王继雪,孙延鸣.盘羊杂交羊SP-A基因克隆与真核表达[J].石河子大学学报(自然科学版),2018,36(3):309-313.
[7]张晓丽,高宝德,刘海燕,等.巴什拜羊重组SPLUNC1蛋白对体外培养的绵羊肺炎支原体生长的影响[J].中国预防兽医学报,2016,38(05):361-365.
[8]冷青文,李志远,鲁海富,等.盘羊体内绵羊肺炎支原体的分离和鉴定[J].中国预防兽医学报,2014,36(3):197-200.