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ABSTRACT: To construct a prokaryotic expressing plasmid for recombinant immunotoxin which fused anti-EGFR scFv together with gelonin toxin, express and verify its function.
The gene fragments coding anti-EGFR single chain fragment were amplified with PCR and cloned into pET32a vector which contains gelonin toxin. The new plasmid was transformed into BL21 (DE3) cells. The induced inclusion bodies were denatured, refolded and purified through SP Sepharose Fast Flow Column. The purified immunotoxin rEG was identified by western blot analysis, and the bioactivity was identified using cell immnuohistochemistry and MTT assay.
The expressing vector pET32a-rEG has been constructed correctly, confirmed by restriction endonuclease digestion and sequencing. The recombinant immunotoxin rEG was purified after denaturing the inclusion bodies, refolding and cationic exchange chromatograph. The purified protein rEG had the right immunology specificity, rEG could efficiently target to EGFR positive cells identified by cell immnuohistochemistry. And the result of MTT assay showed rEG could specifically kill EGFR positive cells.
The recombinant immunotoxin rEG with high purity and biologic activity was prepared in this study, which would become the basic for the further study of the biologic function of rEG.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 09/2011; 42(5):621-4.
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Yan Zhang,
Jiong Li,
Chang-Yong Liu,
Xi-Kun Zhou,
Ji Qiu,
Yin-Bing Zhang, Nong-Yu Huang,
You Li,
Xiang-Jun Chen,
Xiao-Lei Li,
Yong-Sheng Wang,
Han-Suo Yang,
Xian-Cheng Chen,
Bing Kan,
Yong-Qiu Mao,
Hong-Xin Deng,
Li Yang,
Yan-Jun Wen,
Xia Zhao,
Yu-Quan Wei
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ABSTRACT: Psoriasis is a chronic and relapsing inflammatory skin disease associated with various immunologic abnormalities. Repeated subcutaneous injection of interleukin-4 (IL-4) has been established as an effective treatment to counteract psoriasis.
We investigated whether gene therapy using IL-4 expression plasmid (pIL-4) via transdermal delivery was an alternative treatment for psoriasis. In our experiment, dimethylsulfoxide (DMSO) was used as a penetration enhancer.
At first, the penetration efficiency of the complex of reporter plasmid accompanied by DMSO was investigated both in vitro and in vivo. Then, the antipsoriasis efficiency of the treatment with pIL-4-DMSO was tested in mice.
The expression of the reporter gene was detected in epidermis and dermis both in vitro and in vivo. More importantly, the psoriasis symptoms were relieved, and significant reductions in some psoriasis-associated factors were observed after pIL-4-DMSO treatment.
We conclude that the topical application of pIL-4-DMSO can treat psoriasis to a significant extent.
Dermatology 08/2010; 221(1):84-92. · 2.05 Impact Factor
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ABSTRACT: To construct Recombinant Mouse Interleukin 4 prokaryotic expressing plasmid, express it in E. coli strain BL21 (DE3), purify and identify the expressed cytokine.
The optimized mIL-4 cDNA fragment was cloned into the prokaryotic expressing vector pET-32a (+) to generate pET32/rmIL-4 and transformed into BL21 (DE3) cells. After induction, the expressed protein wasfound to be in the inclusion of E. coli cells. The induced product was purified through Q Sepharose Fast Flow Column and Gel Filtration Column under renaturing condition. The purified protein was identified by Western blot analysis, and the biologic activity was identified by the generation of mIL-4 dependence cell CTLL-2 and in vivo experiment of mouse psoriasis model.
The recombinant plasmid pET32/rmIL-4 has been constructed correctly. The inclusion body was washed with 3 mol/L guanidine hydrochloride and denaturized in 7 mol/L guanidine hydrochloride. Then, the denaturized protein was gradient dialysis in the condition of pH 9. 5. The protein we purified has the right immunology specificity and biologic activity.
The recombinant mouse interleukin-4 with high purity and biologic activity was prepared in this study,which will become the basis for the further study of the biologic activity of IL-4.
Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition 07/2009; 40(4):579-83.
