Study On The Expression Of Mice Interferon-γ And Bombyxin Ⅱ Using An Endophytic Bacillus Megaterium PE1 Isolated From Potato

As the development of life science and technology plants could provide people not only natural products by the cultivation but also pharmaceuticals, industrial proteins, and secondary metabolites by transgenic technology. Plants could accumulate the foreign proteins in certain organisms such as seeds and tubers, so that facilitate the extraction and purification of foreign proteins. The fact that plants usually provide abundant biomass and have high biosynthetic capacity permits the production of foreign proteins on a large scale and at competitive costs. While the methods of transforming and stable expression of foreign gene are limited for it is always complicated and the foreign gene sometimes might be deleted silenced in the plant. Researchers are trying to develop simple, convenient and low-cost methods for transforming and expressing foreign genes. Endophyte was found exist widely in plants. The endophyte-plant host relationship is a balanced symbiotic continuum, ranging from mutualism through commensalism to parasitism. All of our vegetal food consists of endophytes which indicate that endophyte is safe for people. So it would be a efficient pathway to use the transgenic endophyte to express foreing gene in the host plant. Researchers have expressed some foreign protein in plant by the endophyte, while the products were mainly toxic proteins to insects and the prokaryotic expression systems like Escherichia coli and Bacillus megaterium are always used for the expression in vitro. In this study an endophytic Bacillus megaterium strain PE1 was isolated from potato(Solanum tuberosum) tubers and was used to express the foreign genes. Interferon-γ gene of Mus musculus(mIFN-γ) and bombyxin Ⅱ gene from Bombyx mori(BBX-Ⅱ) were transduced into Bacillus megaterium PE1. The transformed PE1 was used to inoculate tissue culture potato. The expression and activity of the two foreign proteins were detected. The main purpose of this research was to study the feasibility of utilizing the simple and convenient prokaryotic transforming methods to express foreign protein in plant so that to bypass the complicated process of plant transformation. We hope to construct an “endophyte-host plant” expression system. The main results and conclusions presented in this thesis are as follows:(1) Endophytic bacteria were isolated from potato tubers and then identified and selected.First, 4 kinds of surface sterilization methods(sterile water, 75% alcohol, 0.1% HgCl and NaClO) were compared for their influence on the isolation of endophytic bacteria from potato tubers. NaClO method was found the best effect among these methods. The endophyte in potato tubers were isolated and cultured on LB plate. Total 27 endophytic bacteria strain were isolated and purified from potato tubers and numbered as PE1-PE27. The 16 S rRNA gene of each strain was amplified by PCR protocol and then sequenced. The sequencing results were blast in RDP and GenBank database. The phylogenetic trees were constructed according to the blast results. The 27 strain belonged to 7 species of 4 genuses among which Bacillus consists of 17 strains, Pseudomonas consists of 5 strains, Staphylococcus consists of 3 strains and Brachybacterium consists of 2 strains. Bacillus strains were 63% of all and showed the majority of endophytes in potato tuber. Since there’re 10 strain of Bacillus belonging to B. megaterium which has been used as the expression system of foreign proteins and showed obvious advantages, the strain PE1 of Bacillus megaterium(named Bacillus megaterium PE1) was selected as the expression system and transducer of foreign genes.(2) Influences of lysozyme concentration, digesting temperature and digesting time on the protoplast formation and regeneration of Bacillus megaterium PE1 were studied.The mono-factor test showed that the influence of each factor on protoplast formation rate(FR) must be opposite to the influence on regeneration rate(RR). So the square of FR×RR was used to evaluate the comprehensive influence of each factor and the orthogonol test was designed and carried out to decide the best treatment conditions. The results of orthogonol test showed that the sequence of three factors’ influence was: temperature > time > concentration of enzyme. The best condition of preparation of Bacillus megaterium PE1 protoplast was: lysozyme concentration 4mg/m L, temperature at 30℃, and digesting time 90 min.(3) mIFN-γ was transformed to Bacillus megaterium PE1 and expressed in PE1 and inoculated potato.cDNA of mIFN-γ was cloned and used to construct the recombinant pHIS1522/mIFN-γ which was transformed into Bacillus megaterium PE1. The expression of m IFN-γ was induced by D-xylose. The results of both SDS-PAGE and ELISA showed mIFN-γ was expressed in Bacillus megaterium PE1 at a concentration of 224.6229mg/L. The Bacillus megaterium PE1 which could express mIFN-γ was used to inoculate potato tissue culture seedlings. Detection of m IFN-γ was carried out 5d, 10 d, 15 d and 20 d after inoculation respectively and the results showed that m IFN-γ existed in the inoculated tissue culture seedlings of all 4 periods, which showed the fact that the transformed Bacillus megaterium PE1 has the ability to infect and host in potato and then express the active foreign protein.(4) BBX-Ⅱ gene was transformed to Bacillus megaterium PE1 and expressed in PE1 and inoculated potato.cDNA of BBX-Ⅱ was cloned and the recombinant pHIS1522/BBX-Ⅱwas constructed and then transformed into Bacillus megaterium PE1. The expression of BBX-Ⅱ was induced by D-xylose. The results of SDS-PAGE showed BBXⅡ was expressed in Bacillus megaterium PE1. The Bacillus megaterium PE1 which could express BBXⅡ was used to inoculate potato tissue culture seedlings. Detection of BBXⅡ was carried out 5d, 10 d, 15 d and 20 d after inoculation respectively and the SDS-PAGE results showed that BBXⅡ was expressed in the inoculated tissue culture seedlings, which showed the fact that the transformed Bacillus megaterium PE1 has the ability to infect and host in potato and then express the active foreign protein.(5) BBXⅡ expressed in Bacillus megaterium PE1 was purified and its activity was detected.BBXⅡ expressed in Bacillus megaterium PE1 was purified by affinity chromatography. Certain amount of purified BBXⅡ was injected into silkworm and mice to test its activities. The results showed that BBXⅡ could decrease the level of trehalose and increase the activity of trehalase in the blood of normal, fasting and hyperglycemic silkworm larva. BBXⅡ expressed in Bacillus megaterium PE1 was also found to have the ability to lower the blood glucose level of normal and streptozotocin induced diabetic mice.The results of this study indicated that endophytic Bacillus megaterium PE1 could express the foreign genes both in the culture media and in its host plant, and the products were active. So it is feasible to construct a new “endophyte-host plant” expression system. This expression system was simple and low-cost in the construction process and safe for people, and it could spread fast to the major part of host plant and be transmitted to the progeny. Especially for the expression of some oral vaccine and protective protein in digestive tract, this system could express these products in the edible plant which could be taken by people like the normal food. In all this new “endophyte-host plant” expression system showed inspiring prospect of development. How to improve the competitive ability of transformed endophytes and increase the production of foreign protein were important problems for the development of this system. Another vital problem is to find a way to utilize the translation and modification system of host plant to modify the foreign protein so that this expression system would compensate the disadvantage of prokaryotic expression system and become a system having advantages of both prokaryotes and eukaryotes.