Prevention of acute vascular rejection in xenograft

我们构建了一个细胞表面结合形式的液相CRP。构建了因子I （fl-Pl）的细胞膜结合形式。低表达fl-Pl的SEC细胞系对人血清中细胞裂解的抑制作用较弱，而高表达fl-Pl的CHO细胞系对细胞裂解的抑制作用超过60%。构建了C1-INHA表面结合形式的人C1-INH (C1-INH- pl)，用C1-INH- pl对CHO和SEC进行稳定转染后的流式细胞分析显示这些分子的表达水平中等。C1-INH-Pl在CHO细胞上阻断人补体介导的细胞裂解约75%，在SEC细胞上阻断60-65%。合成DAF基因是为了使其密码子适应哺乳动物，特别是猪中更常见的密码子。与野生型DAF相比，合成DAF （sDAF）的转染物中蛋白质产量显著增加，但未检测到mRNA升高。与体外实验数据一致，携带sDAF的小鼠在许多器官中的DAF表达高于野生型DAF。建立了C1-INH-Pl的各种缺失突变体的dafcdna和C1-INH-Pl的各种形式的影响评估。而C1-INH-Pl除delta-1-99AA外，其余缺失突变体均未在细胞表面表达。因此，delta- 1- 99aa C1-INH-Pl和delta-SCR1 DAF分子都可以非常有效地下调异种细胞裂解，但实现方式不同。研究了HSV-1糖蛋白gC （HSV-gC1）分子的补体调控功能。CHO细胞转染除含有预先存在的抗hsv - gc1抗体外，对血清的细胞裂解有显著的抑制作用。然而，SEC转染在所有情况下都显示出明显的细胞裂解抑制作用。

We constructed a cell surface-bound form of a fluid phase CRP. Factor I-PlThe cell membrane-bound form of factor I (fl-Pl) was constructed. An SEC line with a low expression of fl-Pl showed a weak inhibition of cell lysis in human serum, whereas a CHO cell transfectant with a high expression of fl-Pl showed over a 60% inhibition of cell lysis.C1-INHA surface-bound form of human C1-INH (C1-INH-Pl) was constructed Flowcytometric profiles of the stable CHO and SEC transfectants with C1-INH-Pl showed a medium level of expression of these molecules. C1-INH-Pl block human complement-mediated cell lysis by approximately 75% on the CHO cell and by 60-65% on the SEC cell, respectively.The codon-optimization DAFThe DAF gene was synthesized in order to adapt its codons for those which are more frequent in mammals, especially pigs.A significant increase in protein production with no detectable mRNA elevation was observed in the transfectants of synthetic DAF (sDAF), compared with the wild-type DAF. Consistent with the in vitro data, the expression of DAF in the mice which carry sDAF was higher than wild-type DAF in many organs.Assessment of the effect of various forms of C1-INH-Pl and DAFcDNAs of various deletion mutants of the C1-INH-Pl were established. While all deletion mutants of C1-INH-Pl except the delta-1-99AA were not expressed on the cell surface. Consequently, both the delta1-99AA C1-INH-Pl and delta-SCR1 DAF molecules are quite effective in downregulating the xenogeneic cell lysis, but accomplished this in different manners.HSV-gC1The complement regulatory function of a HSV-1 glycoprotein gC (HSV-gC1) molecule was investigated. The CHO cell transfectants showed a significant inhibition in cell lysis by the sera except for those that contained the preexisting anti-HSV-gC1 antibody. The SEC transfectants, however, showed a marked inhibition of cell lysis in all cases.