APRT缺陷型CHO细胞系的建立及其重组蛋白表达能力评价

Development of an APRT-deficient CHO cell line and its ability of expressing recombinant protein

摘要:

中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞，腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸，是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因，验证获得的APRT缺陷型CHO细胞系的生物学特性；构建两种真核表达载体：对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP)，分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池；重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度，并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明，CHO细胞aprt基因成功敲除；获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明，与野生型CHO细胞相比，转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%；特别是长期传代培养时，转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05)；构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。

关键词:中国仓鼠卵巢细胞;腺嘌呤磷酸核糖转移酶;基因敲除;重组蛋白表达

Abstract:

Chinese hamster ovary (CHO) cells are the preferred host cells for the production of complex recombinant therapeutic proteins. Adenine phosphoribosyltransferase (APRT) is a key enzyme in the purine biosynthesis step that catalyzes the condensation of adenine with phosphoribosylate to form adenosine phosphate AMP. In this study, the gene editing technique was used to knock out the aprt gene in CHO cells. Subsequently, the biological properties of APRT-KO CHO cell lines were investigated. A control vector expressed an enhanced green fluorescent protein (EGFP) and an attenuation vector (containing an aprt-attenuated expression cassette and EGFP) were constructed and transfected into APRT-deficient and wild-type CHO cells, respectively. The stable transfected cell pools were subcultured for 60 generations and the mean fluorescence intensity of EGFP in the recombinant CHO cells was detected by flow cytometry to analyze the EGFP expression stability. PCR amplification and sequencing showed that the aprt gene in CHO cell was successfully knocked out. The obtained APRT-deficient CHO cell line had no significant difference from the wild-type CHO cells in terms of cell morphology, growth, proliferation, and doubling time. The transient expression results indicated that compared with the wild-type CHO cells, the expression of EGFP in the APRT-deficient CHO cells transfected with the control vector and the attenuation vector increased by 42%±6% and 56%±9%, respectively. Especially, the EGFP expression levels in APRT-deficient cells transfected with the attenuation vector were significantly higher than those in wild-type CHO cells (P<0.05). The findings suggest that the APRT-deficient CHO cell line can significantly improve the long-term expression stability of recombinant proteins. This may provide an effective cell engineering strategy for establishing an efficient and stable CHO cell expression system.

Key words:Chinese hamster ovary cells;adenine phosphoribosyltransferase;gene knockout;recombinant protein expression