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

Mouse cells isolated from peritoneal cavity were washed and inoculated with RPMI 1640 Media (10% FBS) in 96-well plates for 2 hr at 37℃. Serum was diluted by 0.781%, 1.562%, 3.125%, 6.25%, 12.5%, 25%, 50% and 100% with 1×PBS, 1mM CaCl 2 , and 2mM MgCl 2 . 15 Diluted serum, SARS-CoV N protein (100ng/ml) and E. coli (the ratio to cells was 10:1) were added to each well and incubated for 30 minutes at 37℃. The eluate from pET22b-transformed E. coli was used as a negative control for purified N protein to exclude effects due to bacterial components that may active complement. The reaction was stop and cells were fixed with 10% neutral formalin. Complement C3c depositon was detected with FITC-C3c antibody, and the Immunohistochemistry 35 Postmortem autopsy from the 4 patients died in Huoshenshan hospital was carried out by Dr. Xiuwu Bian under the approval of the hospital ethic committee and the family member of the death. The detailed results will be published elsewhere. Paraformaldehyde-fixed lung tissues was used for paraffin tissue sections and immunohistochemical staining with MBL (Santa Cruz), MASP-2 (Santa Cruz), C4α chain (Santa Cruz), C3 (Santa Cruz) or C5b-9 (Calbiochem) 40 antibodies as described in the instruction manual.