生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
775-778
,共4页
李玲%王婷%梁迎春%张立%冯滢滢%周丽英%冀全博%郭靖%徐小洁%叶棋浓
李玲%王婷%樑迎春%張立%馮瀅瀅%週麗英%冀全博%郭靖%徐小潔%葉棋濃
리령%왕정%량영춘%장립%풍형형%주려영%기전박%곽정%서소길%협기농
人VHL基因%克隆%真核表达%肿瘤细胞
人VHL基因%剋隆%真覈錶達%腫瘤細胞
인VHL기인%극륭%진핵표체%종류세포
von Hippel-Lindau%cloning%eukaryotic expression%cancer cells
目的：构建人抑癌基因VHL的真核表达载体，并验证其对肿瘤细胞生长的影响。方法：采用PCR技术从人乳腺文库中扩增人VHL基因，将其克隆到pXJ-40-myc载体中，酶切和测序验证后转染人胚肾293T细胞，通过蛋白免疫印迹鉴定其表达；转染人乳腺癌ZR75-1细胞和肝癌HepG2细胞，通过CCK8法测定细胞生长曲线。结果：从人乳腺文库中扩增得到约650 bp的DNA片段，并克隆至pXJ-40-myc载体上，且测序与目的序列完全一致；转染人胚肾293T细胞后，蛋白免疫印迹检测到相对分子质量为26×103的目的基因表达产物；细胞生长曲线显示，转染myc-VHL的乳腺癌、肝癌细胞较空载体细胞生长慢。结论：构建了myc-VHL真核表达载体，myc-VHL抑制癌细胞生长，为进一步研究VHL在肿瘤发生发展中的功能奠定了基础。
目的：構建人抑癌基因VHL的真覈錶達載體，併驗證其對腫瘤細胞生長的影響。方法：採用PCR技術從人乳腺文庫中擴增人VHL基因，將其剋隆到pXJ-40-myc載體中，酶切和測序驗證後轉染人胚腎293T細胞，通過蛋白免疫印跡鑒定其錶達；轉染人乳腺癌ZR75-1細胞和肝癌HepG2細胞，通過CCK8法測定細胞生長麯線。結果：從人乳腺文庫中擴增得到約650 bp的DNA片段，併剋隆至pXJ-40-myc載體上，且測序與目的序列完全一緻；轉染人胚腎293T細胞後，蛋白免疫印跡檢測到相對分子質量為26×103的目的基因錶達產物；細胞生長麯線顯示，轉染myc-VHL的乳腺癌、肝癌細胞較空載體細胞生長慢。結論：構建瞭myc-VHL真覈錶達載體，myc-VHL抑製癌細胞生長，為進一步研究VHL在腫瘤髮生髮展中的功能奠定瞭基礎。
목적：구건인억암기인VHL적진핵표체재체，병험증기대종류세포생장적영향。방법：채용PCR기술종인유선문고중확증인VHL기인，장기극륭도pXJ-40-myc재체중，매절화측서험증후전염인배신293T세포，통과단백면역인적감정기표체；전염인유선암ZR75-1세포화간암HepG2세포，통과CCK8법측정세포생장곡선。결과：종인유선문고중확증득도약650 bp적DNA편단，병극륭지pXJ-40-myc재체상，차측서여목적서렬완전일치；전염인배신293T세포후，단백면역인적검측도상대분자질량위26×103적목적기인표체산물；세포생장곡선현시，전염myc-VHL적유선암、간암세포교공재체세포생장만。결론：구건료myc-VHL진핵표체재체，myc-VHL억제암세포생장，위진일보연구VHL재종류발생발전중적공능전정료기출。
Objective: To construct the eukaryotic expression vector of myc-tagged human von Hippel-Lindau (VHL) gene and examine activity of its product in cancer cells. Methods: Human VHL gene was amplified from human mammary cDNA library, and was inserted into eukaryotic expression vector of pXJ-40-myc, followed by en?zyme digestion and sequencing confirmation. The resulting plasmid was transfected into HEK293T cells, and its ex?pression was detected by Western blotting. Effect of myc-VHL on cancer cell growth was investigated by CCK8 as?say, using breast cancer and liver cancer cells. Results: The DNA fragment about 650 bp was amplified by PCR, and verified by sequencing after it was cloned into pXJ-40-myc. Western blotting showed the expression product of myc-VHL with Mr of 26 kD in the HEK293T cells. Cell growth curve demonstrated that cells transfected with myc-VHL grew significantly slower than those transfected with empty vector. Conclusion: The eukaryotic expres?sion vector of myc-VHL was constructed, and its product played suppressive role on cancer cell growth, based on which, VHL in cancer development and progression will be studied.
