一、發(fā)表文章轉(zhuǎn)染簡(jiǎn)述：
文章使用美國Zeta Life公司,#Advanced DNA RNA轉(zhuǎn)染試劑（#AD600150，Zeta Life, USA) 將構(gòu)建的pCMV-GSDMD-N-HA 載體質(zhì)粒DNA（圖 6A）轉(zhuǎn)染到原代奶牛子宮內(nèi)膜上皮細(xì)胞(BEECs)， 48小時(shí)后用Western blotting檢測(cè)GSDMD，并檢測(cè)到蛋白表達(dá)確定轉(zhuǎn)染成功（圖 6B）。
重建細(xì)胞炎癥模型，提取總蛋白，GSDMD表達(dá)及其裂解的N使用蛋白質(zhì)印跡法檢測(cè)末端蛋白。結(jié)果表明 BEEC 暴露于 LPS（10 和 30 µg/ml）24 小時(shí)可以裂解 GSDMD 并導(dǎo)致焦亡（圖 6C）
文章標(biāo)題LPS Mediates Bovine Endometrial Epithelial Cell Pyroptosis Directly Through Both NLRP3 Classical and Non-Classical Inflflammasome Pathways。
發(fā)表文章單位：中國農(nóng)業(yè)科學(xué)院畜牧與藥學(xué)研究所，農(nóng)業(yè)農(nóng)村部獸藥開發(fā)重點(diǎn)實(shí)驗(yàn)室，蘭州研究所。
二、轉(zhuǎn)染原代上皮細(xì)胞(BEECs)部分論文欣賞
1、LPS Exposure for 24 h Leads to BEEC Inflflammation and Cleaved GSDMD to Pyroptosis
We constructed a pCMV-GSDMD-N-HA vector (Figure 6A) and transfected it to BEECs to overexpress GSDMD, and detected protein expression withWestern blotting after 48 h to ensure the transfection was successful (Figure 6B). The cell inflflammation model was rebuilt,
total proteinwas extracted, and GSDMD expression and its cleaved N terminal protein were detected using Western blotting. The resultsshowed that BEEC exposed to LPS (10 and 30 µg/ml) for 24 h could cleave GSDMD and lead to pyroptosis (Figure 6C).
2、Genetic Recombination
Genetic Recombination Technology was adopted to ligate bovine GSDMD DNA fragments into the ampicillin-resistant pCMV-HA vector and transform it into E. coli. Brieflfly, the bacteria were grown on an LB agar medium containing ampicillin sodium for 24 h at 37°C. A single colony was picked and added to the LB liquid medium containing ampicillin sodium and culturedfor 16 h at 37°C with shaking at 300×g. Plasmid DNA was extracted with an Endo[1]free Plasmid Mini Kit I and quantifified using a Polluton100+. The Zeta Life Transfection Kit was used to transfect the plasmid into BEECs, and LPSwas used to construct the cell inflflammation model. The cell total protein was extracted and detected by an HA tag and Western blotting to evaluate the GSDMD protein expression and its cleavage during cell pyroptosis. The vector construction, restriction endonuclease cleavage verifification, and sequencing verifification were done by Genecreate Biological Company。
三、Zeta Life 公司與美國加利福尼亞大學(xué)舊金山校區(qū)聯(lián)合開發(fā)用于哺乳動(dòng)物細(xì)胞、活體動(dòng)物轉(zhuǎn)染的 Advanced DNA RNA 第三代多肽小分子轉(zhuǎn)染試劑，此技術(shù)成為新的蛋白功能、免疫細(xì)胞及干細(xì)胞治療、研發(fā)及生產(chǎn)的主要關(guān)鍵技術(shù)之一。
