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       嘌呤霉素（Puromycin）是由白黑鏈霉菌（Streptomycesalboniger）發(fā)酵代謝產(chǎn)生的一種氨基糖苷類抗生素，通過抑制蛋白質(zhì)合成而殺死革蘭氏陽性菌，各種動物和昆蟲細(xì)胞。某種特殊情況下有效作用大腸桿菌。作用機(jī)制在于嘌呤霉素是氨酰-tRNA分子3´末端的類似物，能夠與核糖體的A位點(diǎn)結(jié)合并摻入到延伸的肽鏈中。嘌呤霉素同A位點(diǎn)結(jié)合后，不會參與隨后的任何反應(yīng)，從而導(dǎo)致蛋白質(zhì)合成的提前終止并釋放出C-末端含有嘌呤霉素的不成熟多肽。

       嘌呤霉素產(chǎn)生菌Streptomycesalboniger內(nèi)發(fā)現(xiàn)的pac基因編碼嘌呤霉素N-乙酰轉(zhuǎn)移酶（PAC），賦予機(jī)體對嘌呤霉素產(chǎn)生抗性。這一特性如今普遍應(yīng)用于篩選特定攜帶pac基因質(zhì)粒的哺乳動物穩(wěn)定轉(zhuǎn)染細(xì)胞株。

       嘌呤霉素在細(xì)胞穩(wěn)轉(zhuǎn)株篩選中的普遍應(yīng)用與慢病毒載體的特性有關(guān)，現(xiàn)在商業(yè)化的慢病毒載體多數(shù)都攜帶pac基因。在某些特定情況下，嘌呤霉素亦可以用來篩選轉(zhuǎn)化攜帶pac基因質(zhì)粒的大腸桿菌菌株。

本產(chǎn)品在常溫下穩(wěn)定，避免高溫和酸堿環(huán)境。對多種革蘭氏陽性菌和革蘭氏陰性菌有效，特別是對鏈球菌、肺炎球菌等有強(qiáng)效。對哺乳動物細(xì)胞也有一定毒性。

研究應(yīng)用：

1. 作為抗生素篩選工具，用于研究細(xì)菌耐藥性。
2. 用于細(xì)胞培養(yǎng)中防止細(xì)菌污染。

使用說明

1. 建議使用濃度

哺乳動物細(xì)胞：1-10 μg/mL，最佳濃度需要?dú)缜€來確定。

大腸桿菌：LB瓊脂培養(yǎng)基篩選穩(wěn)定轉(zhuǎn)化pac基因的大腸桿菌，使用濃度為125 μg/mL。

【注】：使用嘌呤霉素篩選大腸桿菌穩(wěn)轉(zhuǎn)株需要精確的pH值調(diào)節(jié)，而且受宿主細(xì)胞本身的影響。

2.溶解方法

用蒸餾水溶解嘌呤霉素配制成50 mg/mL的母液，經(jīng)0.22 μm濾膜過濾除菌后分裝于-25~-15℃凍存；也可溶于甲醇，配制成10 mg/mL的儲存液。

3. 嘌呤霉素殺滅曲線的確定（以shRNA轉(zhuǎn)染或者慢病毒轉(zhuǎn)導(dǎo)為例）

嘌呤霉素有效篩選濃度跟細(xì)胞類型、生長狀態(tài)、細(xì)胞密度、細(xì)胞代謝情況及細(xì)胞所處細(xì)胞周期位置等有關(guān)。為了篩選到穩(wěn)定表達(dá)的shRNA細(xì)胞株，確定殺死未轉(zhuǎn)染/轉(zhuǎn)導(dǎo)細(xì)胞的最低濃度嘌呤霉素至關(guān)重要。建議初次做實驗的客戶一定要建立適合自身實驗體系的殺死曲線（kill curve）。

1）Day 1：24孔板內(nèi)以5~8×104 cells/孔的密度鋪板，鋪足夠量的孔以進(jìn)行后續(xù)的梯度實驗。37℃細(xì)胞孵育過夜。

2）Day 2：①準(zhǔn)備篩選培養(yǎng)基：含不同濃度嘌呤霉素的新鮮培養(yǎng)基（如0-15 μg/mL，至少5個梯度）；②往孵育過夜后的細(xì)胞內(nèi)更換新鮮配制的篩選培養(yǎng)基；之后37℃孵育細(xì)胞。

3）Day 4：更換新鮮的篩選培養(yǎng)基，并觀察細(xì)胞存活率。

4）根據(jù)細(xì)胞的生長狀態(tài)，約2-3天更換新鮮的篩選培養(yǎng)基。

5）每日監(jiān)測細(xì)胞，觀察存活細(xì)胞率，從而確定抗生素篩選開始4-6天內(nèi)有效殺死非轉(zhuǎn)染或所有非轉(zhuǎn)導(dǎo)細(xì)胞的藥物最低濃度。

4. 哺乳動物穩(wěn)定轉(zhuǎn)染細(xì)胞株的篩選

等轉(zhuǎn)染含有pac基因的質(zhì)粒后，細(xì)胞在含有嘌呤霉素的培養(yǎng)基中增殖，以篩選出穩(wěn)定轉(zhuǎn)染子。

1）細(xì)胞轉(zhuǎn)染48 h后，將細(xì)胞（原樣或稀釋）置于含有適當(dāng)濃度嘌呤霉素的新鮮培養(yǎng)基中培養(yǎng)。

【注】：當(dāng)細(xì)胞處于分裂活躍期時，抗生素作用最明顯。細(xì)胞過于密集，抗生素產(chǎn)生的效力會明顯下降。最好進(jìn)行細(xì)胞分盤使其密度不超過25%。

2）每隔2-3天，移除和更換含有嘌呤霉素的培養(yǎng)基。

3）篩選7天后評估細(xì)胞形成的病灶。病灶可能需要額外的一周或者更多時間，這依賴于宿主細(xì)胞系和轉(zhuǎn)染篩選效率。

【注】：每日進(jìn)行細(xì)胞生長狀態(tài)的觀察。嘌呤霉素的篩選至少需要48 h，有效濃度嘌呤霉素的篩選周期一般在3-10天。

4）轉(zhuǎn)移和放置5-10個抗性克隆到一個35 mm的培養(yǎng)皿中，用選擇培養(yǎng)基繼續(xù)培養(yǎng)7天。此次富集培養(yǎng)是為日后的細(xì)胞毒性實驗做準(zhǔn)備。

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-25~-15℃保存，有效期2年。

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