HRCA and application in detection of genetically modified plant / 生物工程学报

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.

Study of the expression membrane protein LASS2 / 生物工程学报

A human membrane protein LASS2 (Homo sapiens longevity assurance homologue 2 of yeast LAG1), which has important physiologic functions, was expressed in three different expression systems. Only the LASS2 protein carboxyl terminal hydrophilic fragment could be expressed in the prokaryote expression system and its polyclonal antibody was produced. The full length of LASS2 protein could be expressed successfully in both eukaryotic in vitro translation system and Bacuvirus expression system: Bac-to-Bac system. SDS-PAGE analysis revealed that the molecular weight of expressed product of LASS2 was about 28 kD. The product was also proved by Western blot. This recombinant LASS2 protein was purified by metal affinity resin and the purity is above 90%.