目的构建毕赤酵母基因工程菌,以实现朱黄青霉α-1,6-葡聚糖酶的异源高效表达。方法人工合成朱黄青霉HI-4的α-1,6-葡聚糖酶基因ZHdex,克隆到毕赤酵母分泌型表达载体pPIC9K,电转入毕赤酵母GS115。甲醇诱导目的蛋白表达。结果成功在毕赤酵母GS115中对ZHdex基因进行表达,SDS-PAGE表明重组毕赤酵母在66×103附近有目的蛋白表达,与理论值一致。在摇瓶水平,甲醇诱导培养120 h后,α-1,6-葡聚糖酶的最高酶活达28.79 U/mL,蛋白质浓度为0.56 mg/mL。结论获得一株重组毕赤酵母GS115-ZHdex,其产物具备α-1,6-葡聚糖酶活性,成功地实现朱黄青霉α-1,6-葡聚糖酶的异源高效表达。

Objective To construct the recombinant Pichia pastoris to heteroexpress the α-1,6-dextranase from Penicillium minioluteum. Methods α-1,6-Dextranase gene (ZHdex) of Penicillium minioluteum was synthesized and cloned into vector pPIC9K, then transformed into the expression host P. pastoris GS115 via electroporation. Methanol was used for protein expression. Results ZHdex was successfully expressed, and the Mr of recombinantα-1,6-dextranase was estimated to be 66×103, which is consistent with the theoretical value. In shaking flasks, the highest α-1,6-dextranase activity was determined to be 28.79 U/mL with a protein concentration of 0.56 mg/mL after 120 h induction with methanol. Conclusion A recombinant P. pastoris GS115-ZHdex can be obtained and its product can possessα-1,6-dextranase activity. Theα-1,6-dextranase from Penicillium minioluteum can be successfully heteroexpressed.

β-甘露聚糖酶已经广泛的在毕赤酵母中异源表达,为了提高β-甘露聚糖酶在毕赤酵母中的产量,本文研究了培养温度对β-甘露聚糖酶在毕赤酵母中表达的影响.通过比较不同温度下β-甘露聚糖酶在转录和蛋白表达水平的差异,发现降低温度虽然降低了mRNA表达量,但是外源蛋白得到积累增加;降低培养温度可以显著提高重组菌株的活性,从而减少细胞死亡带来的胞内蛋白酶的释放,最终使得低温下发酵液中的蛋白酶活力显著下降.研究结果表明降低培养温度能显著提高β-甘露聚糖酶在毕赤酵母中的表达量.

β-Mannanase has been extensively expressed heterologously in Pichia pastoris. The effect of the culture temperature on the expression ofβ-mannanase in Pichia pastoris was investigated in order to improve the production ofβ-mannanase in Pichia pastoris. Through comparing the difference of transcription and protein expression levels ofβ-mannanase under different culture temperature, it’s found that mRNA level decreased with decreasing of the temperature, but exogenous protein was accumulated. Lowering culture temperature could evidently improve the cell aviability of the recombinant strain, which could reduce the release of intracellular protease from the death cell, ultimately make the protease activity in the fermentation broth decrease significantly under the low temperature cultivate condition. The research results show that lowering culture temperature can evidently improve the expression ofβ-mannanase in Pichia pastoris.

为了在毕赤酵母X-33中表达犬防御素-3,并检测其抗菌活性,根据毕赤酵母密码子偏好性,人工合成犬防御素-3基因,构建重组质粒PpiczɑC-CBD-3,线性化后电转至毕赤酵母菌X-33,经抗性筛选,阳性克隆用10mL/L甲醇诱导表达,表达产物经Tricine-SDS-PAGE电泳分析,并用琼脂空穴扩散法测定其抗菌活性。表达产物经Tricine-SDS-PAGE检测,在分子质量8.5ku左右有目的条带,与预期大小一致,表达的犬防御素经琼脂空穴扩散法检测显示出一定的抗菌活性。研究表明,犬防御素-3在毕赤酵母中成功分泌表达,为进一步研究奠定了基础。

The rescarch aimeds at expressing the canineβ-defensin-3 gene in Pichia pastoris and determine-ing the antimicrobial activity of expressed product;According to the bias codon of Pichia pastoris ,the ca-nineβ-defensin-3 gene was synthesized,and the recombined plasmid PpiczɑC-cbd-3 was constructed ;the linearized recombined plasmid was transformed into Pichia pastors ;X-33 by electroporation,the positive clones were resistantly screened and induced by 1.0% methanol.The expressed product was identified by Tricine-SDS-PAGE,and determined for antibacterial activity by agarose diffusion methods.A protein band about 8.5 ku was detected,and consistent with the purpose bands.Agarose diffusion test showed the ca-nineβ-defensin-3 had relatively antibacterial abilities.It showed that the canineβ-defensin-3 was successful-ly expressed in Pichia pastoris ,which laid a foundation for further study on defensins.

目的：利用毕赤酵母系统表达戊型肝炎病毒（HEV）ORF2编码蛋白第452～605氨基酸多肽（p154），探讨毕赤酵母表达系统用于研发戊型肝炎基因工程疫苗的可能性。方法：采集戊型肝炎患者粪便，进行RT-PCR检测和测序鉴定，并依此为模板扩增ORF2 p154基因，克隆到表达载体pPICZαA上，电转化入毕赤酵母宿主菌GS115，经筛选鉴定后将携带目的基因的重组克隆菌进行诱导表达，对表达产物进行SDS-PAGE和蛋白质印迹法分析。结果：利用基因重组技术构建了含HEV p154片段的pPICZαA重组质粒，并在毕赤酵母菌株GS115中实现了分泌性表达，表达的目的蛋白p154分子质量约为22 kDa，上清中p154表达量可达到200μg· ml -1。 p154可与HEV感染兔恢复期血清及HEV单克隆抗体6F9进行反应，表明酵母表达的p154具有良好的抗原性。结论：HEV ORF2 p154在毕赤酵母表达系统中得到了成功表达，为进一步研发真核表达的戊型肝炎基因工程疫苗奠定了实验基础。

Objective: To investigate a new expression system to develop recombinant hepatitis E vaccine . Methods:The recombinant plasmid was transformed into GS 115 by electroporation .The transformants were cultured in selection media and screened for the existence of foreign gene by PCR .Then the positive transformant was induced and the expression product was detected by SDS-PAGE and Western blotting assays .Results: The p154 gene of HEV was cloned into the Pichia pastoris expression vector pPICZαA.The p154 could be secreted from yeast and its molecular weight was 22 kDa,in supernatant the p154 was accumulated up to 200μg· ml -1 .The result of Western blotting demonstrated that the p 154 could be specifically recognized by monoclonal antibody against HEV and the recovery serum of rabbit infected by HEV .Conclusion:The successful expression of HEV p 154 protein in Pichia pastoris provides foundation for the further development of recombinant vaccine against hepatitis E .

为大量获得具有生物活性的大黄鱼生长激素(Pseucdosciaena crocea,Growth Hormone,pcGH),利用本实验室分离的天然大黄鱼生长激素基因在毕赤酵母表达系统中的表达进行了研究。把pcGH克隆到酵母整合型质粒载体中构建重组表达载体pPIC9K-pcGH,电击转化将pcGH转化进his4缺陷型毕赤酵母GSll5菌株染色体上,经甲醇诱导得以高效表达,SDS-PAGE证实了表达产物为大黄鱼生长激素,表达量约为62.5mg/L。

This study aimed at high-yield expression of the pcGH gene which was separated from natural large yellow croaker in the yeast Pichia pastoris in order to produce amount of large yellow croaker growth hormone (Pseucdosciaena crocea, Growth Hormone, pcGH) with the feature of biological activity for further use. The pcGH gene was first cloned into the integrative expression vector of methylotrophic yeast P. Pastoris to form the recombinant vector pPIC9K-pcGH, and the linearized recombinant plasmid was then transformed into the chromosome of the HIS4 mutant yeast GS115 by electroporation, and it could finally get high-yield expression induced by methanol. SDS-PAGE confirmed that the expression product was pcGH, and it led to a level as high as of 62.5 mg/L.