【作者】 王丽娜；

【导师】 朱平； 程克棣； 孔建强；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 青蒿素是目前治疗疟疾的首选药物,但天然来源受到多种因素制约,通过基因工程方法生产其中间体青蒿酸、进而化学半合成青蒿素是解决资源问题的重要途径之一。为构建青蒿酸高产酵母工程株系,本论文开展了紫穗槐-4,11-二烯（简称:紫穗槐烯,AD）的生物合成途径工程研究并取得如下进展:1.ADS整合型酿酒酵母工程菌的构建:根据同源双交换的原理,构建可以在酿酒酵母的λDNA序列处进行双交换的紫穗槐-4,11-二烯合酶（ADS）基因（ADS）的整合载体。λDNA序列在酿酒酵母基因组中大约有200个拷贝,把外源基因整合在这个位点可以大大提高外源基因在酿酒酵母中表达的拷贝数。构建ADS整合载体首先要把酿酒酵母的强启动子ADH和终止子连在载体上,然后把ADS基因亚克隆到强启动子ADH和终止子之间,再连接一个营养缺陷型筛选标记基因URA3,还要把λDNA序列分成两个交换臂连在所有待整合基因的两端。用这个构建好的载体转化营养缺陷型酿酒酵母,筛选出ADS整合型工程菌。得到的菌株经过发酵培养后,用GC-MS检测发酵产物,经过计算,其中一株工程菌产紫穗槐烯的量达到117.0mg·L^-1。2.ADS整合型工程菌和ADS附加体型工程菌AD产率比较:将本论文构建的ADS整合型工程菌和实验室以前构建的ADS附加体型工程菌各选3株,同时发酵培养,然后进行发酵液提取和GC-MS检测。结果显示,整合型工程菌的AD产率明显高于附加体型工程菌的AD产率（P＜0.01）。3.鲨烯合酶基因（SQS）反义RNA表达载体的构建:设计反义RNA时,人们普遍应用由翻译起始位点向上下游延伸0.5kb—3.0kb的片段作为重组质粒的逆向插入序列,5′和3′非翻译区（UTR）往往具有良好的效果。本研究把SQS mRNA的4个基因片段:SQS1（5′-UTR）,SQS7（3′-UTR）,SQS3*4（5′UTR加5′编码区）以及SQS5*6（3′-编码区加3′-UTR）分别反向插入到pYeDP60的GAL10/CYC1半乳糖诱导型启动子的下游,构建成4个相应的SQS反义RNA表达载体pYeD/SQS1、pYeD/SQS7、pYeD/SQS3*4、pYeD/SQS5*6。4.SQS反义RNA表达对AD酿酒酵母工程菌生物合成的影响:用上述4个SQS反义RNA表达载体分别转化ADS整合型酵母工程菌,得到含有SQS反义RNA的ADS酵母工程菌。用GC-MS检测发酵产物中的AD和鲨烯（SQ）的量,初步研究发现,转入SQS反义表达载体后,鲨烯（SQ）和AD的产率比转入前均有所下降。可能的原因是:所分析的重组菌样本量比较少,而这些重组菌中转入的反义基因片段除了引起SQS表达下调以外,还引起ADS表达的部分抑制。本论文还开展了重组SARS未知功能小蛋白的大肠杆菌表达、纯化及抗体制备研究:5.SARS-CoV未知功能小蛋白的表达、纯化、鉴定及抗体制备:在大肠杆菌BL21trxB（DE3）中,表达了重组SARS-CoV未知功能小蛋白X4、X5和ORF10。对X5和ORF10重组蛋白进行了Ni⁺亲和层析纯化。ORF10纯化时,用低浓度咪唑(60mM·L^-1)梯度洗脱,获得22KD和44KD两个蛋白条带;在用高浓度咪唑(120mM·L^-1)梯度洗脱时,只有分子量大小为22KD的蛋白条带。制备这两种条带并送中国科学院生物物理研究所进行LC-MS/MS测定,结果显示:在22KD和44KD两种蛋白样品中均能检测到与ORF10蛋白序列100%同源的若干个片段,两种样品所测得的片段分别占总ORF10蛋白序列理论值的38.3%和45.1%,由此推测:所获得的重组蛋白就是ORF10蛋白,其中44KD的蛋白条带可能是该蛋白的二聚体的形式,这也很可能是病毒进入人体后起作用的形式。在纯化X5蛋白时,用和ORF10一样的纯化方法却没有得到X5蛋白,推测X5蛋白是以包涵体形式存在。用尿素变性溶解包涵体,然后所得上清用亲和柱层析,Tris缓冲液复性的办法得到了高产率、高纯度的蛋白。用复性后的蛋白免疫兔子得到了高效价的X5蛋白的抗血清。Western免疫印迹显示尿素变性纯化再复性的X5蛋白的抗体能够和菌体中表达的X5蛋白结合。以包涵体形式表达的X5蛋白纯化后的产率达到93.3mg·L^-1是以非包涵体形式表达的ORF10蛋白纯化后产率的5倍。更多还原

【Abstract】 Artemisinin is the preferred drug for treating malaria.Because of the limited natural resource,one of the potential methods is firstly to produce the intermediate artemisinic acid by the metabolic pathway engineering,and then to obtain artemisinin by chemical semi-synthesis with artemisinic acid as the substrate.To get the engineered yeast with high-yield of artemisinic acid,we carried out the research on the metabolic pathway engineering of amorpha-4,11-diene in the engineered yeast,and some of the results are presented here:1.Construction of the engineered Saccharomyces cerevisiae that produces amorpha-4,11-diene.According to homologous recombination mechanism,the yeast expression vector was constructed,which harbors the ADS gene ofArtemisia annua and the twoλDNA fragments of yeast.After the ADS gene was introduced into the yeast host, a new biosynthetic pathway of amorpha-4,11-diene was established.The twoλDNA fragments were ligated to the up-and down-streams of the ADS gene,respectively,so that the ADS gene might be inserted in theλDNA sites on the yeast genomic DNA. There are about 200λDNA copies in the yeast genome,and the multi-copies integration of the ADS gene on the yeast genome might greatly improve the amount of ADS in yeast, leading to the increase of the product amorpha-4,11-diene.The procedure is as follows: firstly,the yeast ADH1-promoter and terminator were cloned into the vector,respectively. Then the ADS gene,the auxotrophic marker URA3 and theλDNA sequences were subcloned into the vector step by step.This recombinant plasmid was transformed into the URA-deficient yeast W303A and the engineered yeast was screened on the SD-ura" medium at 28℃.After 3-4 days cultivation,the positive clones were characterized by PCR.The fermentation products of these positive engineered yeasts were detected by GC-MS and the amorpha-4,11-diene yield in one of these strains reached up to 117.0 mg·L^-1.2.Comparison of the AD yield between the episomic plasmid-transformed and the integrative plasmid-transformed yeasts.Comparison of the AD yields between the two kinds of the engineered yeasts was performed as follows:three strains were chosen from each of the engineered yeasts.Their fermentation products were extracted and then detected by GC-MS.The results indicated that the AD yield of the integrative plasmid-transformed yeast was much higher than that of the episomic plasmid-transformed yeast（P＜0.01）.3.Construction of the antisense RNA expression vectors of the yeast squalene synthase.Normally,the DNA fragments with 0.5kb-3.0kb in length,which harbor the translation start coding site,or with the 3’-untranslated regions（UTR） are used for the construction of the antisense RNA expression vectors.In this paper,the four SQS fragments,namely SQSI（5’-UTR）,SQS7（Y-UTR）,SQS3^*4（5’-UTR plus 5’-coding region） and SQS5^*6（3’-coding region plus 3’-UTR）,were used to construct the SQS antisense RNA expression vectors for the SQS antisense RNA transcription in the host yeast after transformation and cultivation.All of the DNA fragments were reversely inserted at the downstream of the galactose-induced promoter GAL10-CYC1 in the vector pYeDP60,leading to the four recombinant vectors:pYeD/SQS1,pYeD/SQS7, pYeD/SQS3^*4,and pYeD/SQS5^*6.4.Effects of the SQS anti-sense RNA expression on the AD biosynthesis of the engineered yeast.The four SQS antisense RNA expression plasmids mentioned above were transformed into the AD producing strain.Products of the transformants were quantified by GC-MS.The results indicated that both the squalene and AD yields were declined and the further analysis is ongoing.Besides,the SARS-CoV recombinant protein work was also carried out.5.Expression,purification,characterization and antibody preparation of SARS-CoV putative unknown proteins.The SARS-CoV putative unknown proteins X4,X5 and ORF 10 were expressed by the E.coli recombinants,respectively.ORF 10 and X5 proteins were purified on a Ni⁺ affinity chromatography.With the low concentration (60mM·L^-1) of imidazole washing elution,two protein bands（22 KD and 44 KD） of the ORF10 transformant were observed by SDS-PAGE.But when the imidazole concentration increased(120raM·L^-1),only one protein band（22 KD） was obtained. Both the 22KD and 44 KD protein bands on SDS-PAGE gel were cut off for the LC-ESI-MS/MS analysis.It showed that the detected sequences of the 22KD band match 38.3%of the ORF 10 protein sequence with 100%identity,while those of the 44KD band match 45.1%of the ORF 10 protein sequence with 100%identity.This result implied that the ORF 10 protein can be expressed in E.coli in the dimmer form.The recombinant X5 protein was expressed in the form of inclusion body.The inclusion body was dissolved in high concentration of urea.The supernatant was purified by Ni⁺ affinity chromatography,and then the denatured protein was refolded in a series of gradient solutions of urea.The purified protein was obtained with the purity of＞95% and the yield of 93.3mg·L^-1（five times of the non-inclusion protein ORF10）. Polyclonal antibody of this protein was obtained,and Western blot assay indicated that the antibody could specifically bind to the X5 protein produced by the recombinant strain..更多还原