Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao

Binding of MASP-2 to MBL was assessed by ELISA. As mentioned above, Nunc Maxisorb plates were coated with 10 μg mannan per well in 100 mM Na 2 CO 3 /NaHCO 3 (pH 9.6) at room temperature overnight and blocked with 2% HSA. MBL protein (1 μg/ml) was incubated in 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl 2 , 100 μg/ml HSA, and 0.5‰ TritonX-5 100 at 4°C for 2 hr. Purified MASP-2 and N (or control) proteins were added to the wells at different times to obtain final concentrations of 0.2 mg/ml and 200 ng/ml, respectively. The plates were washed after 32 hr of incubation at 4°C, and the binding of MASP-2 were detected with anti-MASP-2 antibody followed by HRP-conjugated rabbit anti-goat antibody. The enzyme activity of HRP was detected using TMB incubation for 30-60 min at RT, and the reaction was 10 stopped with 2 M H 2 SO 4 . The OD was measured at 450 nm using a microplate reader.