WO2016095326A1 - 一种och1缺陷型抗cd20四价抗体表达毕赤酵母的构建方法 - Google Patents

一种och1缺陷型抗cd20四价抗体表达毕赤酵母的构建方法 Download PDF

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WO2016095326A1
WO2016095326A1 PCT/CN2015/071605 CN2015071605W WO2016095326A1 WO 2016095326 A1 WO2016095326 A1 WO 2016095326A1 CN 2015071605 W CN2015071605 W CN 2015071605W WO 2016095326 A1 WO2016095326 A1 WO 2016095326A1
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och1
gene
pichia pastoris
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ura3
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李云森
朱婷婷
王丽萍
和运
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北京集智新创科技有限公司
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  • the invention belongs to the technical field of genetic engineering, and particularly relates to a method for constructing a Pichia pastoris strain of an OCH1 gene-deficient anti-CD20 tetravalent antibody.
  • Pichia pastoris expression system is one of the most successful exogenous protein expression systems. Compared with other existing expression systems, P. pastoris processes, excipates, post-translational modifications and sugars in expression products. The basic modification has obvious advantages and has been widely used for the expression of foreign proteins.
  • Pichia pastoris is not suitable for the expression of glycoproteins, and its expression product is a high mannose type, which is easy to produce an immune response in human body, and limits the use of Pichia as a host strain.
  • the presence of ⁇ -1,6-mannosyltransferase encoded by the OCH1 gene is an important cause of high mannose formation.
  • the CD20 antigen is a B cell differentiation antigen. Since CD20 is only expressed in pre-B lymphocytes, immature B lymphocytes, mature B lymphocytes, and activated B lymphocytes, there is no CD20 expression in plasma cells, lymphoblasts, and other tissues in human serum. There is also no free CD20 present. Therefore, CD20 can be a good therapeutic target for the treatment of B lymphoma.
  • a preparation method of an anti-CD20 tetravalent antibody is disclosed in the patent CN101544694A, but since the expression vector used is Escherichia coli, it may contain endotoxin and affect its application as a pharmaceutical protein.
  • the Pichia pastoris X-33 genome was used as a template to clone the upstream and downstream homologous sequences of the OCH1 gene, and the upstream and downstream homologous sequences of the OCH1 gene were fused to the gene och1, and the open reading frame of the OCH1 gene was deleted from the fusion gene;
  • the URA3 obtained in the fusion gene och1 and 2) obtained in 1) was introduced into the Pichia pastoris vector pPICZ ⁇ A to construct a pPICZ ⁇ A-URA3-och1 plasmid;
  • the pPICZ ⁇ A-URA3-och1 plasmid was transformed into Pichia pastoris strain JC308, and the positive clone ⁇ och1-strain was obtained by genome screening and phenotypic screening;
  • step 2) the fusion gene obtained in step 1) was cloned into the Pichia pastoris expression vector pPIC9 to form a recombinant expression vector pPIC9 (C 2 B 8 (ScFvHL) 2 Fc);
  • the recombinant expression vector obtained in the step 2) was used to transform the Pichia pastoris engineering ⁇ och1, and the positive clone was screened.
  • the Pichia pastoris ⁇ -1,6-mannosyltransferase (OCH1) gene is knocked out, which prevents the formation of high-mannose-type sugar chains. Reduce the difference in glycosylation between yeast-expressed proteins and human natural proteins to ensure the safety of pharmaceutical proteins.
  • Figure 2 is a URA3 gene clone
  • Figure 3 is a wild type OCH1 gene clone
  • Figure 4 is a clone of the left arm (5' end) and the right arm (3' end) of OCH1;
  • Figure 6 is a construction of a CD20 expression plasmid
  • Figure 7 is a result of pPIC9 (C 2 B 8 (ScFvHL) 2 Fc) ligation
  • Figure 8 is the result of ⁇ och1/CD20 protein expression.
  • a method for constructing an OCH1-deficient anti-CD20 tetravalent antibody expressing Pichia pastoris comprising the steps of:
  • OCH1-5F Two pairs of nucleotide primers OCH1-5F, OCH1-5R, OCH1-3F, OCH1-3R were designed according to the Pichia pastoris OCH1 gene sequence reported in Genebank (E12456).
  • the X-33 strain genome was used as a template to clone the homologous arm sequences at both ends of OCH1.
  • OCH1-5F the product of the OCH1-5R clone SEQ ID NO: 10
  • OCH1-3F the sequence of the product of the OCH1-3R clone is SEQ ID NO: 11
  • Figure 4 OCT1 left arm (5' end) and right arm (3' end) clone electrophoresis map, the reading frame sequence of about 1,200 bp is deleted between the homologous arms on both sides, the fusion fragment och1 is about 1600 bp in size, and the sequence is SEQ ID NO :12, an open reading frame in which the OCH1 gene is deleted in the fusion gene.
  • the primer sequences are as follows:
  • sequence is SEQ ID NO: 1
  • OCH1-3F the sequence is SEQ ID NO: 3
  • OCH1-3R sequence as SEQ ID NO: 4
  • the primer sequence was searched for Pichia pastoris URA3 gene Genbank (AF321098), the amplified sequence of this gene encodes a protein region (CDS sequence), and the URA3 gene CDS region sequence sequence is SEQ ID NO: 13, and the size is 838 bp fragment.
  • Figure 2 is an electropherogram of the URA3 gene clone.
  • FIG. 1 is a construction diagram of the OCH1 knockout vector
  • the plasmid pPICZ ⁇ A-URA3-och1 was linearized by BstEII, electroporated to transform Pichia pastoris JC308, and cultured on MD+His+Arg+Ade+Sorbitol plate for 30 to 5 days at 30 °C. The yeast genome was correctly verified. The second reorganization is correct and a second reorganization can be carried out.
  • the recombinant strain was picked up, inoculated into YPD medium, and cultured in a shake flask at 200 rpm for 10 hours at 26 ° C.
  • the bacterial solution was applied to MD+His+Ura+Arg+His+Sorbitol+5FOA plate, and cultured at 26°C for 5 days. .
  • Two YPD plates were planted on a single colony on a plate, and cultured at 26 ° C and 38 ° C, respectively, were able to grow in the former and could not grow colonies in the latter as possible OCH1-deficient strains.
  • P1: sequence is SEQ ID NO: 7
  • P2: sequence is SEQ ID NO: 8
  • the positive clone ⁇ och1 strain was obtained by genome screening and phenotypic screening
  • Figure 6 is a map of the construction of a CD20 expression plasmid.
  • the fusion gene C 2 B 8 (ScFvHL) 2 Fc sequence of the C 2 B 8 single-chain antibody ScFv gene and the human antibody IgG 1 Fc was designed according to the human anti-CD20 mAb sequence information disclosed in US Pat. No. 6,399,061. Electropherogram of the fusion gene C 2 B 8 (ScFvHL) 2 Fc.
  • the sequence of the C 2 B 8 (ScFvHL) 2 Fc is SEQ ID NO: 9
  • the sequence of the C 2 B 8 (ScFvHL) 2 Fc translated amino acid is SEQ ID NO: 14
  • step 2) the fusion gene obtained in step 1) is cloned into Pichia pastoris expression vector pPIC9 to form a recombinant expression vector;
  • the pPIC9 (C 2 B 8 (ScFvHL) 2 Fc) plasmid was linearized by StuI and electroporated to convert ⁇ och1. Clones that were positive for genome validation were recorded as ⁇ och1/CD20.
  • Figure 8 is the result of ⁇ och1/CD20 protein expression.
  • the post-fermentation uses a 2L water jacket stainless steel fermenter (with oxygen supply, pH micro-control controller, dissolved oxygen, agitation, temperature and nutrient feed, and electronic foam control), fermented base salt contained in the 1L medium of the fermenter And 4% glycerol, which were sterilized at 122 ° C for 30 minutes, followed by sterile addition of 2.4 ml / L trace salt (PTM1) and 100 mL of the starting seeded cells (BMGY culture). Stirring was maintained at 800 rpm, temperature was 30 ° C, oxygen was 30%, pH 5.0, ammonium hydroxide 25%.
  • PTM1 2.4 ml / L trace salt
  • the glycerol equilibration period lasts for 24 hours until the glycerol is exhausted, and then the glycerol supply period is continuously added with 50% (w/v) glycerol and 12 mL/LPTM1, and the rate of the trace salt is 18 ml/h/l until the glycerol supply concentration reaches 100-200 g. /L.
  • 12 mL/L PTM1 was supplied at a rate of 2 g/h/L for 18 hours, 4 g/h/L, 3 hours or 6 g/h/L for 3 hours, and finally 6-7 g/h/L for 5 minutes. 6 days until the end of the fermentation.
  • the cells were collected and centrifuged at 2,500 x g for 10 minutes at 4 ° C to extract and purify the protein of interest.

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Abstract

提供了一种OCH1缺陷型抗CD20四价抗体表达毕赤酵母的构建方法，首先将菌株JC308的编码α-1，6-甘露糖转移酶的OCH1敲除，将改造后的无表达能力的och1与筛选标记连入pPICZαA中，导入野生型毕赤酵母JC308，经同源重组后筛选得到OCH1缺陷型菌株，即Δoch1，然后将合成的抗CD20四价抗体的序列与毕赤酵母表达质粒pPIC9相连，导入OCH1基因缺陷菌株Δoch1中获得该抗CD20四价抗体表达菌株。

Description

一种OCH1缺陷型抗CD20四价抗体表达毕赤酵母的构建方法 技术领域
本发明属于基因工程技术领域，具体涉及一种OCH1基因缺陷型抗CD20四价抗体表达毕赤酵母菌株的构建方法。
背景技术
毕赤酵母表达系统，是目前最为成功的外源蛋白表达系统之一，与现有的其它表达系统相比，巴斯德毕赤酵母在表达产物的加工、外分秘、翻译后修饰以及糖基化修饰等方面有明显的优势，现已广泛用于外源蛋白的表达。但是，毕赤酵母不适于糖蛋白的表达，其表达产物为高甘露糖型，容易在人体中产生免疫反应，限制了毕赤酵母作为宿主菌的使用。OCH1基因编码的α-1，6-甘露糖转移酶的存在，是导致高甘露糖形成的重要原因。对OCH1基因的敲除，可有效阻断酵母对糖蛋白的翻译后高甘露糖化修饰，得到的糖蛋白为低甘露糖结构，可规避毕赤酵母高甘露糖修饰弊端，同时为毕赤酵母后续糖基化改造作基础。
CD20抗原是一种B细胞分化抗原。由于CD20仅在前-B淋巴细胞、未成熟B淋巴细胞、成熟B淋巴细胞、激活B淋巴细胞中表达，而在浆细胞、淋巴多能干细胞以及其它组织均无CD20的表达，在人体血清中亦无游离CD20的存在。因此CD20可作为B淋巴细胞瘤治疗的一个很好治疗靶点。专利CN101544694A中公布了一种抗CD20四价抗体的制备方法，但由于其使用的表达载体是大肠杆菌，可能含有内毒素而影响其作为医药蛋白的应用。
毕赤酵母作为真核细胞具有与人体细胞相似的N-糖基化代谢途径，但是毕赤酵母和人的N-糖基化过程还存在一定差异。毕赤酵母的N-糖基化修饰为高甘露糖型N-糖基化(Heavy mannose type)，而人体细胞中的N-糖基化修饰主要为复杂型N-糖基化(Complex type)和杂合型N-糖基化(Hybrid type)。毕赤酵母中产生的高甘露糖型的N-糖基化修饰，一方面会改变药用糖蛋白的活性和反应动力学特征，另一方面进入人体后可能导致过敏反应。因此，未经改造的毕赤酵母，并不适合于大部分医药用糖蛋白的生产。
目前绝大半部分医药类蛋白都是利用哺乳动物细胞生产的。然而，因为哺乳动物表达体系本身还存在生产成本过高、产量偏低、培养过程中容易受到污染等问题，限制了其在医药蛋白领域的大规模应用。为了解决这一问题，研究人员较早的就开始寻求在大肠杆菌和芽孢杆菌等 原核微生物中表达这些蛋白。但原核生物由于缺乏对真核生物蛋白质的复性功能、缺乏对真核生物蛋白质的修饰加工系统、内源性蛋白酶降解空间构象不正确的外源蛋白、细胞周质内含有种类繁多的内毒素等特点为医药蛋白的生产带来了诸多不便。采用同属真核细胞并拥有较强分泌蛋白能力的毕赤酵母作为医药蛋白表达体系，是解决当前蛋白难以实现低成本、批量化生产的最佳选择。
发明内容
本发明要解决的技术问题是克服现有的缺陷，提供一种OCH1基因缺陷型抗CD20四价抗体表达毕赤酵母菌株的构建方法：包括以下步骤：
一、重组菌Δoch1OCH1菌株的构建
1)、构建融合成基因och1
以毕赤酵母X-33基因组为模板，克隆OCH1基因的上游和下游两段同源序列，将OCH1基因的上游和下游两段同源序列融合成基因och1，所述融合基因中缺失OCH1基因的开放阅读框；
2)、克隆乳清酸核苷-5-磷酸脱羧酶基因URA3；
3)、将1)中所得融合基因och1和2)中所得URA3导入毕赤酵母载体pPICZαA，构建得到pPICZαA-URA3-och1质粒；
4)、将pPICZαA-URA3-och1质粒转化毕赤酵母菌株JC308，经基因组筛选和表型筛选，得到阳性克隆Δoch1-菌株；
二、抗CD20四价抗体表达菌株的构建
1)、构建C2B8单链抗体ScFv基因与人抗体IgG1Fc的融合基因C2B8(ScFvHL)2Fc
克隆CD20单抗C2B8单链抗体ScFv基因和人抗体IgG1Fc基因；将两个C2B8单链抗体ScFv基因串联后并与人抗体IgG1Fc基因连接，得到融合基因C2B8(ScFvHL)2Fc；
2)、将步骤1)中得到的融合基因克隆到毕赤酵母表达载体pPIC9，构成重组表达载体pPIC9(C2B8(ScFvHL)2Fc)；
3)、用步骤2)中得到的重组表达载体转化毕赤酵母工程菌Δoch1，筛选阳性克隆。
目前国内已实现CD20单抗在真核表达系统的表达。中信国建在利用CHO细胞实现了CD20单抗及抗CD20四价抗体的真核表达，体外实验(CDC及ADCC)和体内小鼠荷瘤实验都取得了较好的效果。但是由于真核细胞培养成本高，操作复杂易污染；而毕赤酵母因其具有强启动子(AOX)，可进行高密度培养，培养成本较低在外源蛋白生产方面占有优势。而目前利用酵母作为表达系统进行药用蛋白生产在国内没有相关的研究。
作为人源蛋白的生产载体，毕赤酵母必须解决蛋白后修饰环节与哺乳动物细胞的差异。α-1，6甘露糖基转移酶存在于毕赤酵母内质网，作用是将甘露糖添加到Man8GlcNAc2寡糖链的α-1，3连接的甘露糖臂上而形成Man9GlcNAc2糖链结构。毕赤酵母内源性的其他的甘露糖基转移酶以此糖链作为底物，继续添加甘露糖，形成高甘露糖型结构。因此我们必须干扰酵母细胞糖基化相关酶的表达，本专利中将毕赤酵母α-1，6甘露糖基转移酶(OCH1)基因敲除，可阻止高甘露糖型糖链的形成，从而缩小酵母表达蛋白与人天然蛋白的糖基化差异，保证药用蛋白的安全性。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1是OCH1基因敲除载体的构建示意图；
图2是URA3基因克隆；
图3是野生型OCH1基因克隆；
图4是OCH1左臂(5’端)和右臂(3’端)克隆；
图5是och1融合基因克隆；
图6是CD20表达质粒的构建；
图7是pPIC9(C2B8(ScFvHL)2Fc)连接结果；
图8是Δoch1/CD20蛋白表达结果。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用明和解释本发明，并不用于限定本发明。
实施例
一种OCH1缺陷型抗CD20四价抗体表达毕赤酵母的构建方法，其特征在于，包括以下步骤：
一、阳性克隆Δoch1菌株的构建
1)、构建融合成基因ΔOCH1
根据Genebank(E12456)中报道的毕赤酵母OCH1基因序列设计两对核苷酸引物OCH1-5F，OCH1-5R，OCH1-3F，OCH1-3R。以X-33菌株基因组为模板，克隆OCH1两端的同源臂序列。 OCH1-5F，OCH1-5R克隆的产物SEQ ID NO：10；OCH1-3F，OCH1-3R克隆的产物的序列如SEQ ID NO：11
图4：OCH1左臂(5’端)和右臂(3’端)克隆电泳图谱，两侧同源臂间缺失1,200bp左右的阅读框序列，融合片段och1大小约1600bp，序列如SEQ ID NO：12，所述融合基因中缺失OCH1基因的开放阅读框。
引物序列如下：
OCH1-5F，序列如SEQ ID NO：1
OCH1-5R，序列如SEQ ID NO：2
OCH1-3F：，序列如SEQ ID NO：3
OCH1-3R：序列如SEQ ID NO：4
2)、克隆乳清酸核苷-5-磷酸脱羧酶基因URA3；
引物序列查询毕赤酵母URA3基因Genbank(AF321098)，扩增序列该基因编码蛋白质区域(CDS序列)，URA3基因CDS区序列序列如SEQ ID NO：13，大小为838bp片段。图2是URA3基因克隆的电泳图谱。
U5序列如SEQ ID NO：5
U3序列如SEQ ID NO：6
3)、将1)中所得融合基因och1和2)中所得URA3连入毕赤酵母载体pPICZαA，构建得到pPICZαA-URA3-och1质粒；图1是OCH1敲除载体的构建图；
4)、将pPICZαA-URA3-och1质粒转化毕赤酵母菌株JC308
质粒pPICZαA-URA3-och1经BstEII线性化后，电击转化毕赤酵母JC308，在MD+His+Arg+Ade+Sorbitol平板上30℃下培养3～5天，提取酵母基因组验证正确的即为第一次重组正确，可进行第二次重组。
挑取发生一次重组菌株，接种YPD培养基中，26℃下200rpm摇瓶培养10小时，菌液涂布于MD+His+Ura+Arg+His+Sorbitol+5FOA平板中，26℃下培养5天。将在平板上生长单菌落分别点种两块YPD平板，分别在26℃与38℃培养，能在前者生长而不能在后者生长菌落为可能的OCH1缺陷株。
基因组验证阳性的克隆记作Δoch1，验证引物如下：
P1：序列如SEQ ID NO：7
P2：序列如SEQ ID NO：8
经基因组筛选和表型筛选，得到阳性克隆Δoch1菌株；
二、抗CD20四价抗体表达菌株的构建
图6是CD20表达质粒的构建图谱。
1)、根据美国专利6,399,061公开的人抗CD20单抗序列信息，设计C2B8单链抗体ScFv基因与人抗体IgG1Fc的融合基因C2B8(ScFvHL)2Fc序列，图7是融合基因C2B8(ScFvHL)2Fc的电泳图谱。C2B8(ScFvHL)2Fc的序列如SEQ ID NO：9，C2B8(ScFvHL)2Fc翻译的氨基酸的序列如SEQ ID NO：14
克隆CD20单抗C2B8单链抗体ScFv基因和人抗体IgG1Fc基因；将两个C2B8单链抗体ScFv基因串联后并与人抗体IgG1Fc基因连接，得到融合基因C2B8(ScFvHL)2Fc；
参照国内专利CN 101544694A公布的抗CD20四价抗体资料及序列，委托上海生工生物工程有限公司全基因合成抗人CD20四价抗体的相关序列C2B8(ScFvHL)2Fc，序列详细信息参照核苷酸序列表：
2)、将步骤1)中得到的融合基因克隆到毕赤酵母表达载体pPIC9，构成重组表达载体；
3)、用步骤2)中得到的重组表达载体转化毕赤酵母工程菌JC308/ΔOCH1
pPIC9(C2B8(ScFvHL)2Fc)质粒经StuI线性化后，电击转化Δoch1。基因组验证为阳性的克隆记作Δoch1/CD20。
4)抗CD20四价抗体的诱导表达
挑取阳性菌株Δoch1/CD20于YPD 30℃培养过夜，以1％接种量接种于100mL的BMMY培养基中，培养至OD600＝6.0，收集菌体，接种到BMGY培养基中培养，每隔24小时加甲醇至终浓度为1％进行诱导表达。30℃甲醇诱导发酵60小时后收细胞，用预冷的(4℃)蛋白提取液(5％甘油，1mM PMSF和50mM磷酸盐缓冲液，pH 7.4)洗细胞并重悬，加入玻璃珠用涡旋振荡器振荡裂解蛋白，10 000×g离心30分钟取上清进行SDS-PAGE检测。图8是Δoch1/CD20蛋白表达结果。
后期发酵采用2L水套不锈钢发酵罐(带氧气供应装置，pH值微处理控制器、溶氧，搅拌、温度和营养饲料，和电子泡沫控制)，发酵罐的1L培养基中含有的发酵基底盐和4％甘油，这些都经30分钟，122℃消毒，之后无菌加入2.4ml/L微量盐(PTM1)和100mL的起始接种细胞(BMGY培养)。搅拌维持在800rpm，温度在30℃，氧气30％，pH 5.0，氢氧化铵25％。首先甘油平衡期持续24小时直至甘油耗尽，接着甘油供给期持续加入50％(w/v)甘油和12mL/LPTM1，微量盐的的速率为18ml/h/l直至甘油供给浓度达到100-200g/L。甲醇供给期开始时12mL/L PTM1以2g/h/L的速率供应18小时，4g/h/L，3小时或者6g/h/L，3小时，最终6-7g/h/L持续5到6天直至发酵结束。收取细胞：4℃，2500×g离心10分钟，提取纯化目的蛋白。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (6)

  1. 一种OCH1缺陷型抗CD20四价抗体表达毕赤酵母的构建方法，其特征在于，包括以下步骤：
    一、重组菌株△och1的构建
    1)、构建融合成基因och1
    以毕赤酵母X-33基因组为模板，克隆OCH1基因的上游和下游两段同源序列，将OCH1基因的上游和下游两段同源序列融合成基因och1，所述融合基因中缺失OCH1基因的开放阅读框；
    2)、克隆乳清酸核苷-5-磷酸脱羧酶基因URA3；
    3)、将1)中所得融合成基因och1和2)中所得URA3导入毕赤酵母载体pPICZαA，构建得到pPICZαA-URA3-och1质粒；
    4)、将pPICZαA-URA3-och1质粒转化毕赤酵母菌株JC308，经基因组筛选和表型筛选，得到阳性克隆△och1菌株；
    二、抗CD20四价抗体表达菌株的构建
    5)、构建C2B8单链抗体ScFv基因与人抗体IgG1Fc的融合基因
    克隆CD20单抗C2B8单链抗体ScFv基因和人抗体IgG1Fc基因；将两个C2B8单链抗体ScFv基因串联后并与人抗体IgG1Fc基因连接，得到融合基因；
    6)、将步骤1)中得到的融合基因克隆到毕赤酵母表达载体pPIC9，构成重组表达载体；
    7)、用步骤2)中得到的重组表达载体转化毕赤酵母工程菌△och1，筛选阳性克隆。
  2. 如权利要求1所述的一种OCH1缺陷型抗CD20四价抗体表达毕赤酵母的构建方法，其特征在于，所述筛选阳性克隆△och1菌株的方法为：
    质粒pPICZαA-URA3-och1经BstEII线性化后，电击转化毕赤酵母JC308，在MD+His+Arg+Ade+Sorbitol平板上30℃下培养3～5天，提取酵母基因组验证正确的即为第一次重组正确，可进行第二次重组。
    挑取发生一次重组菌株，接种YPD培养基中，26℃下200rpm摇瓶培养10小时，菌液涂布于MD+His+Ura+Arg+His+Sorbitol+5FOA平板中，26℃下培养5天。将在平板上生长单菌落分别点种两块YPD平板，分别在26℃与38℃培养，能在前者生长而不能在后者生长菌落为可能的阳性克隆△och1菌株。
  3. 一种融合片段och1，序列如SEQ ID NO：12所示。
  4. 一种C2B8单链抗体ScFv基因与人抗体IgG1Fc的融合基因翻译的氨基酸，序列如SEQ ID NO：14所示。
  5. 权利要求1所述的构建方法构建的毕赤酵母菌株。
  6. 权利要求5所述的菌株在发酵生产蛋白中的应用。
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