Establishing Prokaryotic Expression System of Angiotensin-Converting Enzyme 2 (ACE2) gene in pigs

Hang Xiao; Xin-Tian Nie; Xiao-Xia Ji; Shu-ping Yan; Bin Zhu; Yuan-Shu Zhang

The rat anti ACE2 serum was prepared with the best multiple dilution as a single 292 antibody, and the Sheep anti rat IgG-HRP was used as a two antibody. The purified 293 fusion protein, pig heart, spleen, kidney, liver, duodenum, sheep liver, rat liver, rat 294 liver and chicken liver and kidney tissue protein were analyzed by Western blot. The 295 results showed the fusion protein and pig heart. There was a specific binding protein 296 in the size of 100 kDa in the viscera, spleen, kidney, liver and duodenum (Fig. 8) , and 297 the rest did not appear. The results show that the ACE2 polyclonal antibody can 298 identify the fusion protein and the ACE2 protein in the pig tissues, but the ACE2 . CC-BY 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.12.988634 doi: bioRxiv preprint 299 protein in other species can not be combined, that is, the ACE2 polyclonal antibody 300 against the pig's mouse has good immune response and specificity. 301 3.11 Anti ACE2 serum specificity in mice by immunofluorescent 302 The mouse anti ACE2 serum was diluted by multiple 1:600 as one antibody, the 303 fluorescent enzyme labeled Sheep anti mouse IgG-HRP was two resistance, and the 304 final concentration was 100 ng/mL DAPI for 5 minutes. The immunofluorescence 305 analysis of the pig intestinal epithelial cells, as shown in Figure 9 , that the mouse anti 306 ACE2 could specifically identify the ACE on the membrane of the pig small intestinal 307 epithelial cells. The 2 protein, that is, the polyclonal antibody of pig anti mouse ACE2 308 prepared, has better immunoreactivity. 310 As shown in Figure 10 , the proteins extracted from pig spleen, kidney, liver, lung, 311 pancreas, lymph node, stomach, duodenum, jejunum, ileum, colon, rectum and cecum 312 were analyzed by Western blotting, and the rat anti porcine polyclonal antibody was 313 diluted with 1: 3200 as one resistance, and the IgG of sheep resistant to HRP was two.