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Abstract Invariant chain(Ii) is an important immunity molecule for vertebrate and plays a critical role in major histocompatibility complex(MHC)Ⅱ presenting antigen. To study the relation between chicken(Gus gallus) Ii and MHCⅡ molecules, we constructed 4 lentivirus vectors targeting to silence chicken Ii gene and compared their effect. First, we designed and synthesized 4 silencing chicken Ii gene sequences and obtained double-stranded (short hairpin RNA, shRNA) by one-step annealing method, then inserted them into lentivirus vectors pLL3.7 by a double enzyme digestion, respectively. The 4 recombinant vectors were named pLL-Ii-shRNA236, pLL-Ii-shRNA376, pLL-Ii-shRNA527 and pLL-Ii-shRNA539, respectively. Secondly,these vectors were transfected into 293T cells(human renal epithelial cell line transfected with adenovirus E1A (lethal infection gene)), respectively, and the green fluorescent protein expressed in these cells. When the packaged recombinant lentiviruses were injected into 293T cells, their titers were 2.2×107~5.1×107 TU/mL and their infection efficiency in chicken salvolar macrophage HD-11 was over 95%. Finally, a Real-time PCR was used to detect their effect on interfering mRNA of chicken Ii gene in HD-11 cells. The results showed that the interference efficiency of the 4 recombinant lentivirus vectors were 37%、52%、82% and 66%, respectively, compared with the control group of blank vector, in which pLL-Ii-shRNA527 showed peak efficiency. In conclusion, one recombinant lentivirus vector was selected from 4 vectors, which could most efficiently silence chicken Ii gene transfection in the chicken salvolar macrophage HD-11; these results suggested that the shRNA sequence determines the efficiency of recombinant vector in silencing specific gene. All these will provide experimental basis for studying the relationship between Ii and other immune molecules in presenting antigens.
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Received: 18 February 2014
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