人egf基因在突变枯草芽孢杆菌WYBS2001中的转化、分泌表达及其产物功能研究

通过紫外线突变诱导获得了对感染致病菌具有显著抑菌作用的枯草芽孢杆菌突变株WYBS2001。采用PCR技术人工合成了175bp的人表皮生长因子（hEGF)的基因片段，并引入Pst I、Hind Ⅲ酶切位点，起始密码子及pUS186载体信号肽序列CTTAGA。经DNA测试分析，合成的片段与人egf基因序列完全一致。然后将其克隆至枯草杆菌分泌型质粒载体pUS186上构建成重组质粒pUSE，并转化枯草杆菌突变菌株WYBS2001，获得转入egf基因的枯草杆菌生态工程菌WYBS2001T。RIA检测结果表明，WYBS2001T阳性工程菌培养的上清液中可检测到hEGF。含量为7.6ng/ml，如果培养液中添加蛋白酶抑制剂可提高hEGF检测量，通过多代的培养仍然能够稳定地分泌表达hEGF。生物学功能实验表明，分泌的hEGF对人K562体外培养细胞的增殖和生长具有明显生物学活性。对烧伤动物模型的功能性实验观察到，WYBS2001T工程菌制剂对动物的烧伤有明显的治疗作用，该研究表明，微生态基因工程菌有很好的应用前景。

Transforming and Secreting Expression of Human egf in Mutant Strain WYBS2001 of Bacillas and Its Functions

Mutant strain WYBS2001 of B. subtilis with strong anti-pathgenic activity was obtained by mutagenic ultraviolet rays. The gene fragment of Human Epidermal Growth Factor(hegf) of 175 bp was synthesized by PCR and the restriction sites Pst Ⅰ and Hind Ⅲ , original code and the signal sequence CTTAGA of secreting vector pUS186 were induced in the fragment. The DNA sequencing result revealed that the synthesized fragment was identical with that of human egf. Then the biological engineering strain WYBS2001T with human egf was obtained by transforming pUSE which was constructed by cloning egf into the secreting plasmid pUS186, into mutant strain WYBS2001. The result of RIA showed that hEGF can be found in the supernatant of the cultures and its content was 7.6 ng/ml. And the content can be increased if the proteinase inhibitor was added into the medium. After several generations' culturing, WYBS2001T positive engineering strain can still secrete and express hEGF steadily. The result of experiment showed hEGF had biololgical activity of proliferation and growth of human cell K562 in vitro. WYBS2001T engineering strain had obvious effect on healing the burned animals'models. This research showed microecological gene-engineering bacteria has good applying foreground.