Exploration Of Expression And Preparation Of Polyclonal Antibody Of Rat Novel PMP22CD

Objective PMP22CD obtained by in silico cloning and submitted to GeneBank with the GeneBank accession No.634367 and expressed in testis tissue of rat, which is a novel gene of rat. Its function has not been investigated. Through this research, getting enough information about it.Methods Investigating the basic information of PMP22CD through analysis of bioinformatics. Constructing prokaryotic expression mutate recombinant plasmid PET-32a(+)/PMP22CD and expressing fusion protein in Escherichia coli BL21(DE3 Lysis)induced by IPTG. After purifing fusion protein, immunizing rabitt to obtain PMP22CD polycoloned antibody. Investigating PMP22CD expression pattern by RT-PCR. Exploring PMP22CD expression in testis tissure in mRNA level and protein level through in situ hybridization and immunohistochemistry. Constructing eukaryotic expression recombinant plasmid wild and mutate PEGFP-C1/PMP22CD and observing the distribution of fluorescence under lasers copolymerization microscope after transfecting HeLa cells 48h.Results PMP22CD which had four extron was mapped to chromosome 8q22 and cDNA was 910bp in length, encoding protein which was composed of 231 amino acid residues with four transmembrane helix. Phylogenetic tree analysis indicated that PMP22CD protein possessed an independent evolutional direction from prophase. Anti-rabbit polyclonal antibody of PMP22CD was obtained by constructing prokaryotic recombinant plasmid PET-32a(+)/PMP22CD and expressing fusion protein to immunize rabbit. Its titer and specifity were tested by sepharose double diffusion and Western Blot respectively. PMP22CD expressed in testis of rats, which were above 13 months old. The result of in situ hybridization and immunohistochemistry showed that PMP22CD was specifically expressed in Spermatocytes cells, while no expression in ripe spermatid cells. The result of subcelluar location showed that the strong green fluorescence concentrated around the nuclear membrane when wild PEGFP-C1/PMP22CD over expressed in HeLa Cells. In contrast, the green fluorescence suffused the cytoplasm when mutate PEGFP-C1/PMP22CD over expressed in HeLa Cells and distributed throughout the whole cells when PEGFP-C1 over expressed in HeLa Cells.Conclude PMP22CD was specifically expressed in Spermatocytes cells which are above 13 years old. PMP22CD may be located in Endoplasmic Reticulum, and the signal anchoring peptide played a key role in the processes of PMP22CD location. Prepared anti-rabbit polyclonal antibody of PMP22CD and used it in immunohistochemistry and Western Blot.