摘要
头孢菌素类药物(Cephalosporins)是一族β-内酰胺类广谱抗生素,具有抗菌活性强、疗效高、毒性低等特点。生产这类抗生素的前体头孢菌素C(Cephalosporin C,CPC)由顶头孢霉(Cephalosporium acremonium)发酵生产。用基因工程改造的方法获得高产菌种是进行菌种改良的有效途径。
为了方便的进行顶头孢霉菌的基因工程改造,首先必须构建适合在顶头孢霉中进行基因组整合的启动子,以及整合质粒等工具。本文通过PCR的方法,成功克隆了产黄青霉异青霉素N合成酶(IPNS)启动子pcpC_p(1.16kb)、顶头孢霉异青霉素N合成酶(IPNS)启动子pcpC(452bp),经酶切和测序验证,与NCBI上报道的序列一致。这些启动子可以被顶头孢霉的RNA聚合酶识别,用于启动异源基因在顶头孢霉中的表达。
为了制备VHb抗体用于免疫印迹检测,需要获得较高纯度的VHb蛋白,构建了pET28a-vgb重组质粒,并发现vgb基因的第二个氨基酸编码是影响VHb蛋白表达的决定因素。当该位点突变成缬氨酸时几乎不表达VHb蛋白。
为了实现外源基因的在顶头孢霉中的表达,摸索了顶头孢霉原生质体制备和再生的条件。确定了顶头孢霉传代培养基;用于制备原生质体的培养基为改进型蔗糖液体培养基,培养条件为28℃,150rpm摇瓶培养;采用含0.6mol·L~(-1)的NaCl溶液作为渗稳剂的再生培养基进行再生;采用1000unit·mL~(-1)的Lywellzyme酶进行酶解,0.6mol·L~(-1)的NaCl溶液作为渗透压稳定剂,原生质体数可以达到5.86×10~6个·ml~(-1),再生率达41%。
为将vgb基因整合至顶头孢霉基因组中,本文构建了顶头孢霉转化载体pDH25-pcpC-vgb。该质粒以潮霉素B抗性(hph)作为筛选标记。顶头孢霉RNA聚合酶能够识别pcpC启动子和PtrpC启动子,分别启动vgb基因和潮霉素B抗性基因的表达,vgb基因和潮霉素B抗性基因共用TtrpC终止序列。为进一步转化顶头孢霉奠定了基础。
Cephalosporins are a family of theβ-lactam antibiotic which kill bacteria by interference with the synthesis of bacterial cell wall and destruction of cell wall.Due to wide arrange,higher activity and lower toxicity of antibiotics, Cephalosporins have been regarded as the major antibiotics together with Penicillin,since the industrial production during 60~(th) of 20 century.
In the present study,vgb gene was integrated to the chromosome of Cephalosporium acremonium.By suitable promoter,vgb gene was constitutively expressed to improve the transport of oxygen and the growth of Cephalosporium acremonium under hypoxygen.Therefore,the concentration and quality of Cephalosporium acremonium were improved,the cost of fermentation was decreased,and the yield of Cephalosporin C was enhanced, providing new way to the contradiction of the need of oxygen and the limited provision of oxygen by the instruction during the fermentation of Cephalosporium acremonium.
The construction of the transform system of Cephalosporin was necessary for the study of the molecular biology of Cephalosporium acremonium.An efficient tansformation vector was determined by the strength of the promoter of expressing genes.To express extra gene,the promoter should be recognized by the RNA polymerase of Cephalosporium acremonium.Therefore,to construct a transformation vector,the clone of proper sequence of promoter is required.In the present paper,the promoter pcpC_p of IPNS and pcpC(452bp) promoter of IPNS was cloned.By enzyme digestion and sequencing,the cloned promoters were identical to the data in NCBI.The cloned promoters can be recognized by the RNA polymerase of Cephalosporium acremonium and can initiate the expression of extra gene in Cephalosporium acremonium.
In this paper,in order to obtain high yield and purity VHb protein used for antibody production and ELISA,we contructed plasmid pET28a-vgb.We found the second amino acids gene code have the determinants affect of VHb expression.VHb protein can be purified through metal affinity chromatography by His-Tag label structure.
In order to clone and express vgb in Cephalosporium acremonium,we have studied the preparation and regeneration conditions of Cephalosporium acremonium protoplast.We have identified the condition of medium,culture condition,enzyme,and Osmotic stabilizer.Used 1000unit·mL~(-1) Lywellzyme as enzyme,0.6 mol·L~(-1) NaCl solutions as Osmotic stabilizer,the protoplast number can be 5.86×10~6·mL~(-1),Regeneration rate can be 41%.
To integrated vgb gene in the chromosome of Cephalosporium acremonium,we constructed the transformation vector of Cephalosporium acremonium,i.e.pDH25-pcpC-vgb.This plasmid can transform Cephalosporium acremonium and integrate to the chromosome of Cephalosporium acremonium with HygB as selection marker,the RNA polymerase of Cephalosporium acremonium can recognize promoter pcpC and promoter PtrpC,and separately initiate the expression of vgb gene and Hyg B resistance gene.vgb both used TtrpC termination sequence,providing basis for the transformation of Cephalosporium acremonium.
In this study,we have made a good achievement in the genetic engineering of Cephalosporium acremonium,and the research can also afford the foundation of vgb clone and expression in Cephalosporium acremonium.The results are also beneficial to reconstruct other antibiotic-production fungus.
引文
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