Construction Of Prokarvotic Expression System Fused With Polyhedron Fragment And Primary Exploration Of Its Expression Mechanism

Bombyx mori polyhedron (Ph) of Bombyx mori nuclear polyhedrosis virus is a high-expressed protein and easily dissolves in alkali condition (pH10.8),so it is always expressed withforeign protein. It can not only enhance the production of fused protein, but also it can be purifiedby the method of pH gradient and differential centrifugation. And then the target proteins can beobtained by specific proteinases digested. However, recombinant protein fused Ph only dissolvesin alkali condition. When we digest fused protein, it is not easy to harvest lots of target proteinsbecause the pH was not optimum for specific proteinases. We try to find the main area ofpolyhedron gene (ph) that affects the high expression of recombinant EGFP, and found whether itcan reduce the pH to promote the fused proteindissolved.First,we cut out different fragments of ph gene depended on the tertiary structure of Ph.Then the different fragments fused with EGFP were expressed by vector pET-28a. Finally, weobtained three fused proteins named Ph60-EGFP, Ph120-EGFP and Ph180-EGFP respectively. Allof them are high expressed. It was demonstrated that these fused proteins not only have the sameproperties with Ph, which could dissolve in alkali condition(pH10.8) and could be purified by themethod of pH gradient and differential centrifugation, but also by call-backing the pH, thesefused proteins could dissolve in neutral condition and completely be digested by thrombin.Meanwhile, we used these fragments fused with other two membrance proteins MMGT andABP on the expression vector pET-28a. The result proves that foreign protein fused with thesethree fragments also could be high expressed. And fused proteins also could be dissolved inneutral condition by pH gradient and centrifugation. All of results above confirmed that thesethree fragments of Ph have the same properties of high expressed.What’s more, they have a bettersolubility than Ph, which provides a convenient method on purifying and obtaining mass offoreign proteins.This study also investigated ph gene transcription on the RNA level. First, BmN cells wereinfected with the recombinant viruses which were fused with ph gene and without ph generespectively. When the cells were infected, Actinomycin D was added to inhibit the genetranscription. Then, the RNA of cell and virus was extracted in the different periods. mRNAcontent of the exogenous gene was detected by quantitative PCR at each time point. Finally, thehalf-life of recombinant gene mRNA was calculated by the statistical analysis of data. Then the relationship between mRNA half-life and the efficient expression could be further explored. Theresult show that of the virus fused with polyhedron gene, mRNA content increase or mRNAhalf-life is prolonged. However, neither mRNA content nor half-life of mRNA is less than that ofwild ph gene. The reason might be that environmental factors in the expression system are ofgreat help to the stability of the polyhedron mRNA. When fused with polyhedron gene, mRNAstability of the exogenous foreign gene is to be protected or drived by the translation ofpolyhedron, so the gene is highly expressed. It remains to be further studied that how to lead toefficient expression as well as more detailed internal mechanism.