恶性疟原虫多表位人工随机重组疫苗M.RCAg-1的中试规模制备与鉴定

doi:10.3969/j.issn.0258-8021. 2014. 03.010

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摘要中试规模高效制备恶性疟原虫多表位人工随机重组疫苗M.RCAg-1，并对其免疫原性进行鉴定。20 L培养基规模发酵培养M.RCAg-1的工程菌，产物经高压匀浆破碎后，通过镍琼脂糖凝胶FF层析和琼葡糖凝胶G200 HP层析两步分离纯化获得中试M.RCAg-1。将15只BALB/c小鼠随机地平均分为3组（弗氏佐剂组、中试M.RCAg-1与弗氏佐剂配伍组和小试M.RCAg-1与弗氏佐剂配伍组）进行皮下免疫，利用间接Elisa和间接免疫荧光（IFA）技术对3次免疫后小鼠血清中的特异性抗体进行分析。IPTG诱导4 h后发酵结束，菌体产量25 g/L，M.RCAg-1约占菌体总蛋白的24%；经两步柱层析分离纯化后，目的蛋白纯度大于95%，回收率高达53.2%；与弗氏佐剂对照组相比，中试M.RCAg-1和小试M.RCAg-1都能在小鼠体内诱生高水平的特异性抗体应答，其抗体滴度均可达到1:1 024 000。与此同时，两实验组鼠血清抗体的对疟原虫天然抗原都有识别，其识别水平并无差异（P>0.05）。本研究成功地高效制备出多表位人工随机重组疫苗M.RCAg-1，并在小鼠体内证实其具有良好的免疫原性，为随后进行的M.RCAg-1的临床前研究奠定基础。

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	刘新¹韦新桂^{2 &n}

关键词 ：恶性疟原虫, 蛋白疫苗, 中试发酵, 免疫原性

Abstract：The aim of this work is to produce Plasmodium falciparum multiepitope random constructed vaccine M.RCAg-1 efficiently in pilot scale and identify its immunogenicity. M.RCAg-1 producing E.coli was fermented in 20 L culture. After the fermentation products were homogenized by high pressure, the obtained bacteria lysate was purified by Ni FF affinity chromatography and G200 HP gel filtration chromatography. Fifteen BALB/c mice were randomly divided into three groups (the group of Freund’s adjuvant, the group of pilotscale M.RCAg-1 combined with Freund’s adjuvant and the group of labscale M.RCAg1 combined with Freund’s adjuvant) and injected subcutaneously. The serum of these mice was collected after third immunization to analyze the specific serum antibody by indirect ELISA and indirect immunofluoreseence assay (IFA) technology. Fermentation process was finished after 4 h induced by IPTG. The collected wet cell yield was 25 g/L, and the expression level of M.RCAg-1 was about 24%. After twostep purification, the final M.RCAg-1 was over 95% in purity，the yield was 53.2%．Compared with the control group, both the pilotscale and labscale M.RCAg-1 induced high level antibody response in mice and the antibody titter was 1∶1 024 000.At the same time, serum antibody of the two experimental groups recognized the natural antigen of Plasmodium falciparum very well and there was no difference of recognition between the two groups(P>0.05). With the successful production of Plasmodium falciparum multiepitope random constructed vaccine M.RCAg-1 efficiently in pilot scale and the confirmation of its good immunogenicity in mouse model, this study provids the foundation for preclinic study of M.RCAg-1.

Key words： plasmodium falciparum protein vaccine pilot scale fermentation immunogenicity

基金资助:国家高技术研究发展(863计划) (2012AA02A406)

引用本文:

刘新¹韦新桂^{2 &n. 恶性疟原虫多表位人工随机重组疫苗M.RCAg-1的中试规模制备与鉴定[J]. 中国生物医学工程学报, 2014, 33(3): 329-334.

LIU Xin¹WEI XinGui²GAO Yu\|Hui¹WANG Heng^1*. Pilot Scale Production and Identification of Plasmodium Falciparum Multiepitope Random Constructed Vaccine M.RCAg-1. journal1, 2014, 33(3): 329-334.}

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http://cjbme.csbme.org/CN/10.3969/j.issn.0258-8021. 2014. 03.010 或 http://cjbme.csbme.org/CN/Y2014/V33/I3/329

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