National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P. O. Box 577, Faisalabad, 38000, Pakistan.
Mol Biol Rep. 2012 Apr;39(4):4391-400. doi: 10.1007/s11033-011-1227-y. Epub 2011 Sep 22.
The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.
从微生物到哺乳动物表达系统生产干扰素 α，在成本、可扩展性、安全性和真实性方面具有一定的优势。现代生物技术利用转基因作物以具有成本效益的方式获得大量复杂的蛋白质。为了克服从生物安全角度来看的一些挑战，叶绿体转化策略是最佳方法之一，因为在大多数栽培物种中，质体严格地通过母系遗传。在本研究中，干扰素 α5 基因是通过使用复杂的寡核苷酸合成的。在合成基因的序列得到确认后，组氨酸残基与凝血酶蛋白酶位点一起被工程化到合成干扰素 α5 基因的上游。然后，重组片段与叶绿体光诱导启动子 rbcl 连接，并通过顺序克隆将其靶向到质体的反向重复区，通过同源重组的两个事件。通过弹道传递方法获得的假定转基因植物，并通过对轰击叶片的抗生素选择进行筛选，然后进行不同轮次的选择和再生以达到均一性。通过聚合酶链反应和 Southern 印迹分析分析了壮观霉素抗性芽。使用酶联免疫吸附测定技术记录了引入的合成基因的表达。与幼叶和衰老叶相比，发现成熟叶片中干扰素的含量相对较高。
