Rat Alr (orf) And The Difference Between The Size Of The Orf Cloning, Expression And Activity Determination

Objective: To analyze distribution of transcript of long ORF of rALR. To construct prokaryotic expression plasmid of rat augmenter of liver regeneration (rALR) (long ORF) and the difference between the long ORF(We call it X.) and short ORF, express target protein in Escherichia coli. The purified target proteins were used to identify their bioactivities. This will be a basis of further studying biological functions of ALR. Methods: The DNA fragments of rALR (long ORF) were amplified by RT-PCR from total RNA with a pair of primers containing different endonuclease sites and were cloned into pET32a(+). The DNA fragments of X were amplified by PCR from recombinant plasmid pET32a(+)- rALR(long ORF) and were subcloned into plasmids pET32a(+). The recombinant plasmids pET32a(+)-rALR(long ORF) and pET32a(+)-X were transformed into Escherichia coli BL21 (ED3). The target proteins were expressed in their host bacteria under the induction of IPTG. The expressed proteins were purified from the cells broken by ultrasound with Ni2+-NTA column. All purified products were identified by SDS- polyacrylamide gel electrophoresis. The antigenicity of the products were identified by ELISA. The effects of the products on proliferations of QGY in vitro were evaluated by 3H-TdR method. Results: The cDNA fragments of rALR(long ORF) were successfully amplified from total RNA of rat testis, while we had not got the cDNA from total RNA of liver of rat newly born. rALR(198aa)-Thi and X-Thi fusion protein were successfully expressed in Escherichia coli after the construction of recombinant plasmid pET32a(+)-rALR(long ORF) and pET32a(+)-X. Their molecular weight, with 4.1(104 and 2.9(104 respective, was in correspondence with theoretic values, and their expression amounts were up to 20% and 25% of total bacteria proteins respectively. Single band was watched in SDS-polyacrylamide gel electrophoresis after purification with Ni2+-NTA column. But rALR(198aa)-Thi and X-Thi fusion protein could not stimulate proliferation of QGY in vitro. It was demonstrated by ELISA that rALR(125aa)-Thi, rALR(198aa)–Thi and X-Thi fusion proteins could react with the polyclonal antibody against hALR, but the product of BL21-pET32a(+) could not.Conclusion: Distribution of transcript of rALR long ORF is different from rALR short ORF. There is mRNA of rALR long ORF in rat testis while there is none in rat liver. The rALR-Thi and X-Thi fusion protein were expressed efficiently and purified successfully. However, the fusion pronteins couldn't stimulate proliferation of QGY cells in vitro and we suspected it due to the part fused in the protein-thioredoxin.