The Construction And Pilot Scale Preparation Of Fusion Protein GTH

This experiment according to the characteristic that there are a lot of gonadotropin releasing hormone receptors(Gn RHR) and the ablity of Pseudomonas aeruginosa exotoxin A transmembrane(TLD) can transport exogenous transmembrane protein into cells and the nature of hyperthermophilic Archaea histone(Hph A) properties which has high affinity with DNA can form protein-DNA complexes,by means of gene engineering constructs two kinds of expression system "gonadotropin releasing hormone Pseudomonas aeruginosa exotoxin A translocation peptidehyperthermophilic Archaea histone fusion protein(GTH) ", and imports the expression engineering strain BL21(DE3) to induce the expression of GTH fusion protein which has the characteristics combining with sperm, active transmembrane transportation and carrying the DNA, therefore,it can be used as tools to transport DNA in the active into the sperm.At the same time the experiment designes the method of the proteins expression and purification methods and pilot GTH preparation process.By comparing the expression levels of two expression vectors and screening optimal medium, can obtain the engineering bacteria. Which can efficiently express the target soluble protein.Through continuous small scale exploration, sonication and purified by column chromatography, protease digestion, dilution layer different conditions change,getting proven purification methods of the bacteria.Mass production of bacteria in the test, obtaining a large number of relatively high purity target protein for subsequent experiments。The purpose of this experiment is to lay the foundation for an exploration to find an effective solution to sperm as a carrier animal transgenic technology and to provide a simple,efficient and inexpensive method for transgenic animal technology. We base on the characristic that there are a large number of gonadotropin-releasing hormone receptor in the acrosome surface to construct an expression of gonadotropin-releasing hormone- Pseudomonas exotoxin A translocating peptide-super thermophilic archaea histone fusion proteins, And develope a method for forming the fusion protein purification GTH to prepare a certain amount of protein for subsequent application,offering some clues and methods to promote the development of transgenic animal technology and livestock to develope a method for forming the fusion protein purification GTH to prepare a certain amount of protein for subsequent application,offering some clues and methods to promote the development of transgenic animal technology and livestock.