With Autoimmune Hemolytic Anemia Gene Cloning And Expression In Prokaryotes

The genes related with autoimmune hemolytic anemia ( AIHA) were identified with the probe of AIHA patient serum by plaque hybridization in situ. It was demonstrated that CD59 gene and U1 70 kd gene are related with AIHA. Subsequently the sequences of Exon3 and Exon4 of CD59 genes from AIHA patients were determined, but there is not any mutation detected. It mentioned that there must be some antibodies against CD59's protection of cells from the complement. CD59 cDNA (not including the sequence encoding signal peptide) was cloned into the E.coli expression vector pET28b(+) in order to express the recombinant human CD59 (rhCD59). rhCD59 has been isolated and purified from E.coli, and some research on the activities of rhCD59 were performed. The result suggested that rhCD59 has the immunogenicity and could protect cells from dissolvision by complement.Since corynebacteria have a rapid growth velocity, no lyoenzyme of protein, no endotoxin, no inclusion bodies and an easy separating and purifying technique of products,it is significant to express rhCD59 in corynebacteria for medical use. In this study, A cryptic plasmid pXZ608 was isolated from Gram positive Corynebacterium glutamicum 227. Sequence analysis revealed that pXZ608 is a novel plasmid because there is no significant homology of the pXZ608 DNA sequence with the sequences reported. pXZ608 is 5949 bp long with a GC content of 57%. There are six open reading frames (ORF) over 300 bp. Among them, the protein encoded by ORF1 was homological with the replication protein (Rep) of pIJ101/pJV1 family that holds rolling circle replication (RCR). The motifs, single- and double- stranded sequences which is necessary for RCR are found in pXZ608, so it suggested that pXZ608 may belong to RCR family. After deletion analysis, the minimal necessary region for autonomous plasmid replication is located in the 2.14 kb BstEII-SacI fragment (nt 174 to nt 2311), which contains only one putative reading regions--ORF1. pXZ608 was used in constructing the shuttle corynebacteria-E.coli vector. With the vector rhCD59 was successfully expressed in corynebacteria, but more research should be performed to increase the yield.