CD163分子胞外区功能结构域在PRRSV感染PAM细胞中的作用

Roles of the extracellular region domains of CD163 molecule in PRRSV infection to PAMs

目的研究猪CD163分子胞外区功能结构域在PRRSV感染PAM细胞中的作用。方法将实验室保存的PET-CD163-P1、PET-CD163-P2、PET-CD163-P3及PET-CD163-P4重组质粒转化到表达菌BL21(DE3)中,以最佳IPTG诱导表达的时间和最适诱导表达的浓度,大量诱导表达CD163-P1、CD163-P2、CD163-P3和CD163-P4重组蛋白,并用不同浓度的尿素洗涤纯化重组蛋白,然后用紫外分光光度计测定蛋白浓度。用不加血清的1640营养液将各重组蛋白稀释为2.0、1.0、0.5、0.25、0.125 mg/ml 5个不同的质量浓度,再分别与等体积的200个TCID_(50)/0.1 ml PRRSV病毒充分混匀后放置于37℃培养箱作用1 h,后用于感染PAM细胞,补加营养液作用48 h,同时设置单纯PRRSV感染对照组,用已建立的绝对荧光定量PCR方法检测感染细胞中PRRSV的拷贝数,并用Graph Pad Prism 5软件对所得到的数据进行处理。结果 CD163各片段不同稀释度的结构域重组蛋白与PRRSV混合作用感染48 h后,PRRSV的拷贝数均明显低于PRRSV单纯感染PAM细胞组的拷贝数,并且重组蛋白质量浓度越高,竞争结合抑制作用越明显。其中,CD163-P1片段各个质量浓度的结构域重组蛋白的竞争结合抑制作用相比其它片段更明显。结论 CD163各片段不同稀释度的结构域重组蛋白与PAM细胞上的CD163受体胞外区结构域在与PRRSV结合时存在竞争作用,该竞争结合使PRRSV的增殖明显降低,并且CD163-P1结构域片段可能在参与PRRSV感染PAM细胞中发挥主要作用。

This study designed to investigate the roles of the extracellular region domains of CD163 molecule in PRRSV infection to PAMs. The conserved PET-CD163-P1, PET-CD163-P2, PET-CD163-P3 and PET-CD163-P4 recombinant plasmids were respectively transformed into the expression strain BL21 （DE3）. By optimizing the time and the concentration of IPTG inducing, a large number of recombinant proteins CD163-P1, CD163-P2, CD163-P3 and CD163-P4 were expressed. The recombinant proteins were washed by different concentrations of urea for purification, and the protein concentrations were measured by UV spectrophotometer. Adjusting concentrations of each domain protein to 2, 1, 0.5, 0.25, and 0.125 mg/ml, respectively in FBS-free 1640, then mixed with the equal volume viral suspension of 200 TCID50 PRRSV Hn-1/06 strain （10^4.8 TCID50/ml） for 1 h at 37℃, then used to infect PAM cells. Forty-eighty hours after supplementing with 1640 media and 10% FBS, PRRSV copies of infected cells were detected by established absolute fluorescence quantitative PCR method, and the obtained data was analyzed with the GraphPad Prism 5 software. The viral copies of infected cells incubated with mixed viral suspension with different concentration recombinant proteins of CD163-P1, CD163-P2, CD163-P3, CD163-P4 domains were lower significantly than that of PAM cells infected with PRRSV alone, and the higher concentration of CD163 domain proteins was, the stronger competitive inhibitory effect was. For different dilution proteins of each segment of CD163, the competitive inhibitory effect of CD163-P1 fragment domains was more obvious compared with other fragments. In conclusion, the different concentration recombinant proteins of CD163-P1, CD163-P2, CD163-P3 and CD163-P4 domains produce a obvious competitive inhibition with the extracellular region domains of CD163 receptors existing in the PAM cells, and the competitive binding inhibit virus replication on PAM cells significantly, suggesting CD163-P1 fragment domain may plays a major role in the process of PRRSV infection to PAM cells.