Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml),即Lentivirus Expressing Human GLP-1R,是碧云天自行研发的一种可以在大多数哺乳动物细胞(包括原代细胞和干细胞)中通过CMV启动子表达人源GLP-1R蛋白的重组慢病毒。本产品感染细胞后可用于筛选GLP-1R的激动剂以用于治疗糖尿病和肥胖症等。本产品带有bsr/BSD/bls抗性基因,感染细胞后可以使用灭瘟素S (Blasticidin S)筛选稳定表达细胞株。
胰高血糖素样肽-1受体(Glucagon-like peptide-1 receptor, GLP-1R)属于B类G蛋白偶联受体家族(G protein-coupled receptors,GPCRs)。胰高血糖素样肽-1 (Glucagon like peptide-1, GLP-1)是GLP-1R的天然配体,GLP-1与GLP-1R的结合会激活下游信号通路,促进胰岛素分泌并抑制胰高血糖素的释放,从而降低血糖水平。近年来,基于GLP-1研发的胰高血糖素样肽-1受体激动剂(Glucagon-like peptide-1 receptor agonist, GLP-IRA)药物,如Exendin-4、利拉鲁肽(Liraglutide)、司美格鲁肽(Semaglutide)已获批上市治疗2型糖尿病和肥胖等[1]。
内部核糖体进入位点序列(Internal Ribosome Entry Site, IRES)是一段茎环结构序列,在上游启动子的控制下IRES连接的两个基因转录成为一条mRNA,在翻译时IRES可以单独招募核糖体,实现IRES上、下游两个不同基因的分别表达。通常下游基因的表达要相对弱一些。
慢病毒感染细胞后会将目的基因随机整合到基因组DNA上,因而可以长期稳定地表达目的蛋白,并且几乎可以感染所有哺乳动物细胞,在很多不分裂的细胞中也可以长期稳定表达,广泛应用于细胞和动物实验。
本产品表达bsr/BSD/bls抗性基因,因此本产品感染培养的细胞后可以使用Blasticidin S HCl (灭瘟素S) (ST018)筛选稳定表达的细胞株以用于后续研究。
碧云天的Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml)是复制缺陷型慢病毒。其3' LTR的增强子功能发生缺失,形成了自失活(Self-inactivating) 3' LTR,并且5' LRT中的U3区域替换成CMV启动子,在感染普通的细胞后不能进行复制和扩增,从而有效降低了本产品在活体生物中的风险。
碧云天推荐的慢病毒感染不同种类体外培养细胞的MOI值参见下表。
Cell line | Tissue | Cancer/cell type | Species | MOI |
A431 | Epithelial | Carcinoma | Human | 5 |
A549 | Lung | Carcinoma | Human | 5 |
Astrocytes | Nervous system | Primary | Human | 1 |
B16-F10 | Epithelial | Melanoma, metastatic | Mouse | 5 |
BMM | Bone Marrow | Primary | Human | 8 |
BxPC-3 | Pancreas, epithelial | Adenocarcinoma | Human | 10 |
H3255 | Lung | Carcinoma, NSCLC | Human | 10 |
HCT116 | Colon | Carcinoma | Human | 5 |
HeLa | Cervix | Carcinoma, epithelioid | Human | 3 |
HEK293T | Kidney | Tumor | Human | 5 |
Hepa1-6 | Liver | Carcinoma | Mouse | 3 |
HMVEC | Endothelial | Endothelial, microvascular | Human | 100 |
HT-29 | Colon | Adenocarcinoma | Human | 3 |
HUVEC | Umbilicus | Endothelial cells | Human | 100 |
Jurkat | Blood | Leukemia, Acute T cell | Human | 10 |
LLC-1 | Lung | Carcinoma | Mouse | 6 |
LNCaP | Prostate | Carcinoma | Human | 5 |
MM200 | Skin | Melanoma | Human | 5 |
MCF-7 | Breast | Adenocarcinoma | Human | 2 |
MDA-MB-231 | Breast | Adenocarcinoma | Human | 1 |
MM-AN | Skin | Melanoma, metastatic | Human | 16 |
MMC | Breast | Carcinoma | Mouse | 4 |
MRC-5 | Lung, embryonic | Fibroblasts | Human | 1 |
NB4 | Blood | Leukemia, acute promyelocytic | Human | 10 |
PC12 | Adrenal gland | Pheochromocytoma | Rat | 20 |
SKOV-3 | Ovary | Adenocarcinoma | Human | 15 |
U-2 OS | Bone | Osteosarcoma | Human | 5 |
实验室常用的逆转录病毒(Retrovirus)、慢病毒(Lentivirus)、腺相关病毒(AAV)和腺病毒(Adenovirus)的主要特征之间的比较和差别参见下表。具体的特定病毒的一些特征和下表相比可能会有一定差异。
Retrovirus | Lentivirus | AAV | Adenovirus | |
Genome | ssRNA(+) | ssRNA(+) | ssDNA | dsDNA |
Coat | Enveloped | Enveloped | Naked | Naked |
Particle size | 90-100nm | 90-100nm | 20-30nm | 60-90nm |
Genome size | 7-10kb | 9kb | 5kb | 38-39kb |
Genome integration | Yes | Yes | No | No |
Packaging capacity | 2.5-5kb | 2.5-6kb | 2.5-4.5kb | 3-8kb |
Infection tropism | Dividing cells | Dividing and non-dividing cells | Dividing and non-dividing cells | Dividing and non-dividing cells |
Relative Transduction Efficiency | ND | 70% | 70% | 100% |
Expression started | 48-72h | 48-72h | 72-96h | 24-48h |
Expression duration | > 2 months | > 2 months | > 6 months | 3-4 weeks |
Expression level | Medium | Medium | Medium | High |
Immune response | Low | Low | Very low | High |
In vivo safety | Medium | Medium | High | Low |
Titer before concentration (IFU/ml) | 106 | 107 | 1011 | 107 |
Titer after concentration (IFU/ml) | ND | 108 | 0.5-1×1013 | 1010 |
Able to obtain high MOI | No (≤ 10 copies integrated) | No (≤ 10 copies integrated) | Yes | Yes |
Biosafety level | BSL-2 | BSL-2 | BSL-1 | BSL-2 |
本产品的滴度不低于10^8TU/ml,适合细胞实验或活体动物实验。TU, transduction unit,即转导单位。TU通过本产品梯度稀释后感染HEK293T细胞后抽提细胞基因组DNA进行qPCR测定获得,以确定具有生物活性的病毒颗粒数量。MOI (Multiplicity of Infection)是病毒感染细胞时,病毒数量与细胞数量的比值。使用10^8TU/ml的本产品,如果按照5 MOI感染6孔板的细胞,每孔50万细胞计算,每100μl共可以感染4个孔;如果按照5 MOI感染24孔板的细胞,每孔10万细胞计算,每100μl共可以感染20个孔。如果MOI值提高,那么相应可以感染的孔数会减少;如果MOI值下调,那么相应可以感染的孔数会增加。
包装清单:产品编号 | 产品名称 | 包装 |
C4053-100μl | Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml) | 100μl |
C4053-500μl | Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml) | 100μl×5 |
— | 说明书 | 1份 |
-80ºC保存,一年有效。-20ºC保存,1-2个月内有效。4ºC保存,一周内有效。
注意事项:反复冻融会降低病毒滴度,如有必要请在收到本产品后分装保存。分装时必须在冰浴上进行。病毒融解后,如果在一周内使用,可以放置于4ºC,但须注意4ºC存放时间越长,滴度下降越明显。如果-80ºC保存时间超过一年,可能会导致滴度下降,此时建议重新测定病毒滴度。
本产品使用前请仔细阅读附录1《慢病毒使用安全规范》。本产品生物安全等级为Biosafety Level 2 (BSL-2),在按照常规的微生物实验操作要求进行操作(Standard microbiological practices)的基础上,还需要注意限制接触、生物危害提示、显著的警示标识、并制定相应的安全规范。
病毒操作中应注意有效防护,绝对禁止在生物安全柜内有任何皮肤直接暴露的情况。实验完成后,请及时清洗双手。严禁直接接触病毒,如意外接触,请及时用清水冲洗,并适当用70%乙醇对皮肤进行消毒。
任何接触过病毒的材料、试剂、样品,应经消毒处理,可以采用1%的SDS溶液、或84消毒液(1:20)浸泡30分钟以上,或121ºC高压灭菌30分钟。
本产品仅限于专业人员的科学研究用,不得用于临床诊断或治疗,不得用于食品或药品,不得存放于普通住宅内。
为了您的安全和健康,请穿实验服并戴一次性手套操作。
BSL | Agents | Practices | Primary Barriers and Safety Equipment |
Facilities (Secondary Barriers) |
1 | Not known to consistently cause diseases in healthy adults | Standard microbiological practices | ■ No primary barriers required. ■ PPE: laboratory coats and gloves; eye, face protection, as needed |
Laboratory bench and sink required |
2 | ■ Agents associated with human disease ■ Routes of transmission include percutaneous injury, ingestion, mucous membrane exposure |
BSL-1 practice plus: ■ Limited access ■ Biohazard warning signs ■ “Sharps” precautions ■ Biosafety manual defining any needed waste decontamination or medical surveillance policies |
Primary barriers: ■ BSCs or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials ■ PPE: Laboratory coats, gloves, face and eye protection, as needed |
BSL-1 plus: ■ Autoclave available |
3 | Indigenous or exotic agents that may cause serious or potentially lethal disease through the inhalation route of exposure | BSL-2 practice plus: ■ Controlled access ■ Decontamination of all waste ■ Decontamination of laboratory clothing before laundering |
Primary barriers: ■ BSCs or other physical containment devices used for all open manipulations of agents ■ PPE: Protective laboratory clothing, gloves, face, eye and respiratory protection, as needed |
BSL-2 plus: ■ Physical separation from access corridors ■ Self-closing, double-door access ■ Exhausted air not recirculated ■ Negative airflow into laboratory ■ Entry through airlock or anteroom ■ Hand washing sink near laboratory exit |
4 | ■ Dangerous/exotic agents which post high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatments ■ Agents with a close or identical antigenic relationship to an agent requiring BSL-4 until data are available to redesignate the level ■ Related agents with unknown risk of transmission |
BSL-3 practices plus: ■ Clothing change before entering ■ Shower on exit ■ All material decontaminated on exit from facility |
Primary barriers: ■ All procedures conducted in Class III BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suit |
BSL-3 plus: ■ Separate building or isolated zone ■ Dedicated supply and exhaust, vacuum, and decontamination systems ■ Other requirements outlined in the text |
产品编号 | 产品名称 | 包装 |
C4047-100μl | Lenti-CRE-TA-Luc-EF1α-Puro (10^8TU/ml, 报告基因慢病毒) | 100μl |
C4047-500μl | Lenti-CRE-TA-Luc-EF1α-Puro (10^8TU/ml, 报告基因慢病毒) | 500μl |
C4053-100μl | Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml) | 100μl |
C4053-500μl | Lenti-CMV-GLP1R-IRES-BSD (10^8TU/ml) | 500μl |