Examination of dye-protein interaction by gel-permeation chromatography

The interaction of Cibacron blue F3G-A with two therapeutic proteins, recombinant human growth hormone and recombinant human interferon-alpha2b, has been examined by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. The complexes of these proteins with Cibacron blue F3G-A have been isolated, and their absorbance spectra have been registered. The influence of Cibacron blue F3G-A on the oligomeric state of proteins has been investigated. It was found that Cibacron blue F3G-A promotes the generation of interferon-alpha2b dimers at pH 5.0.     

Purification of RhIFN- in Bacteria Bodies with Acetic Acid-Water as Mobile Phase in RPLC

llltrl)(luction. fi. \Rccolllbillallt lllllllall illtcrfcroll- (rlllFN-) is a cylokillc exllibitillg alltiviral, alltiprolifcrativc. alld illllll[1llolllodulatory activitics. alld 11as ccrtaill tllcrZlpclltic value in clillicaltreatlllcllt. Gcllg al all first [ised 11ydropllobic illtcractioll cllrolllatograplly (HIC)sllcccssflllly to scpilr:llc alld pllrif}! rlllFN- silllllllallcollsly ill illclusioll body in olleStep.Several years laier. size cxclllsioll cllrolllalogr;lplly', loll cxcllall…     

Establishment ofChlamydomonas reinhardtii chloroplast expression

An expression vector pACⅢ containing the chimeric gene HAV- VP3P1 and HCV-C gene has been constructed and transferred to C. reinhardtii by the biolistic method. The trans-formants have been identified by PCR, Southern-blotting, Northern-blotting and Western-blotting assays after selecting on resistant medium and incubating in the dark. The results show that the chimeric gene has replaced the chIL gene of C. reinhardtii chloroplast genome and expressed correctly under the control of the C. reinhardtii chloroplast double promoters 5' chlL-5' atpA. The analysis of SDS-PAGE indicates that the expressed protein accounts for 5.31 % of total soluble protein.     

Recombinant LipL32 Protein for Leptospirosis Detection in Indonesia

Leptospirosis is an endemic zoonotic disease in tropical countries. However, detection using the microagglutination test (MAT) as a gold standard is difficult to perform in Indonesia. Therefore, recombinant LipL32 protein (rLipL32) has been studied as an antigen for an ELISA test to detect Leptospirosis. We produced rLipL32 using the pRSET-C vector and expressed it in E. coli BL21 (DE3) competent cells. Under native conditions, we purified 2 mg rLipL32 protein from 500 ml culture. Analysis using western blotting and ELISA showed that serum from the bovine positive MAT serovar Hardjo could recognize pure rLipL32 protein. This result confirms an earlier study that indicates that rLipL32 protein is a good antigen for Leptospirosis detection. The diagnostic assay using rLipL32 is safe because it does not use infectious bacteria as an antigen and because it is easy to perform in every diagnostic laboratory in Indonesia. However, further study is still required for field validation of the rLipL32 assay.     

电击法转移含人凝血因子IX基因重组质粒的影响因素

反转录病毒载体在基因治疗中可能会产生野生型病毒颗粒而引起安全性问题，本文研究含ＦＩＸｃＤＮＡ重组质粒基因治疗血友病Ｂ的可能，构建了不具反转录病毒载体结构的两重组质粒ｐＳＣＩＸＴＮ和ｐＣＩＸＴＮ，前者合ＳＶ４０早期启动子和ｈＣＭＶ启动子共同控制的ＦＩＸｃＤＮＡ，后者仅含ｈＣＭＶ启动于控制的ＦＩＸｃＤＮＡ，它们都含有ＴＫ启动于驱动的ｎｅｏ基因，通过电击法将基因转移到ＰＡ３１７和ＨＴ１０８０细胞，在ＨＴ１０８０细胞中的ＦＩＸ表达量分别为２２０、２１２ｎｇ／（１０６细胞·２４ｈ）通过与ｐＣＭＶＩＸ共转染靶细胞，可以增加人ＩＸ因子表达１．５～３．５倍，显示了用质粒载体转染靶细胞在血友病Ｂ基因治疗中是一条潜在可行的途径．本项研究用自制的电击仪转移ＰＡ３１７和ＨＴ１０８０等细胞，最高的转移效率达１０－３，并探讨了细胞种类，ＤＮＡ量，ＤＮＡ结构，电压，脉冲时间与转移效率及表达量的关系．     

用ELISA双抗体夹心法检测人重组白细胞介素2(rHuIL-2)在酵母菌中表达的研究

采用ELISA夹心法检测人重组IL-2在酵母菌中表达产物的免疫学活性.结果表明,本法具有特异性强、简易、快速和重复性好等特点,尤其对利用工程菌制备及纯化rlL-2过程中能较快地监测出是否有该产物表达.     

毕氏酵母流加发酵过程的比生长速率控制

在已建立的毕氏酵母发酵过程结构模型基础上，进行了甲醇流加阶段恒定比生长速率控制研究。实验结果表明，基于模型的甲醇进料策略可以将细胞比生长速率恒定地控制在设定值；在比生长速率均值相等的情况下，恒定比生长速率进料策略较之恒速进料策略蛋白产量更高。     

重组质粒pPIC9K-pZP3α-hCGβ CTP109～145的构建

目的：为发展多特异性避孕疫苗,制备重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,用基因工程技术构建重组质粒pPIC9K－pZP3α－hCGβCTP^109-145,方法：根据pZP3α全长和hCG 0633CTP109-145编码的DNA序列设计两对引物,通过部分重聚合酶链式反应（overlaping PCR）,扩增出pPIC9K－pZP3α－hCGβCTP^109-145片断,克隆到载体质粒pPIC9K中,然后导入大肠杆菌DH5α,用PCR和双酶切反应鉴定重组质粒,并用DNA测序仪对重组质粒测序,结果：3步PCR扩增出pPIC9K－pZP3α－hCGβCTP^109-145DNA片段,插入到载体质粒pPIC9K的克隆位点,获得重组pPIC9K－pZP3α－hCGβCTP^109-145表达质粒,测序结果显示插入序列与设计预期完全一致。结论：重组质粒pPIC9K－pZP3α－hCGβCTP^109-145构建成功,为表达重组抗原pPIC9K－pZP3α－hCGβCTP^109-145,探讨重组多特异性避孕疫苗抗生育效能的研究建立基础。