Suppression The Expression Of Cap10 Gene Of Cryptococcus Neoformans Cells By Eukaryotic Vector-mediated RNA Interference

Objective:To construct a siRNA expression vector encoding a siRNA targeting cap10 and transfect it into Cryptococcus neoformans cells, and evaluate the inhibition effect on the expression of cap10 on Cryptococcus neoformans.Methods:According to the cap10 cDNA sequence in GenBank, 1 pair of oligo nucleotides was designed and synthesized. After primer annealing, they were inserted into plasmid psilencer4.1-CMV neo to construct the siRNA eukaryotic expression vector. The recombinant vector was transfected into Cryptococcus neoformans cell with chemical method of LiAc. The positive cells were selected by G418. To construct FQ-PCR system for the detection of cap10, specific primers and probes were designed according to the constant sequences of five serological types(A,B,C,D,AD)cryptococcus neoformans, the sequences was amplified by PCR and ligated into pGEM-T Easy vector;then, we appreciated the system,including its sensitivity, specificity and reproducibility. Every group of Cryptococcus neoformans cells were cultivated with YPD and those of total RNA were extracted. The expression of cap10 was detected by FQ-PCR. Every group of Cryptococcus neoformans cell were incubated with murine macrophage-like cell J774A.1 and the phagocytic indexs and ratios were determined by microscopic observation method. And the results were analyzed by SPSS13.0 software. Results:1.Sequencing result of siRNA eukaryotic expression vector targeting cryptococcus neoformans cap10 was same as the designed sequence.2.To extract and sequence the plasmid transfected in positive strains, the result was same as the designed sequence.3.The sensitivity of the FQ-PCR was 104copies/μl, the linear range was 10⁴～10¹⁰ and the CV of inter-assay and intra-assay were 0.31% and 2.72%.4.The average expression of cap10 of Cryptococcus neoformans cells transfected with pS4.1-siRC-1 was 175534.62 copies/μl, was obviously lower than the control experiment group(321995.52 copies/μl) and the blank experiment group(562931.66 copies/μl)(P < 0.05), and the inhibition ratio was 54.51%.5.The average phagocytic ratio and phagocytic index of J774A.1 incubated with Cryptococcus neoformans cells transfected with pS4.1-siRC-1 was 0.100573 and 10.06%, was higher than the control experiment group(0.065676 and 6.46%) and the blank experiment group(0 and 0%) (P<0.05).Conclusions:1. siRNA eukaryotic expression vector targeting cryptococcus neoformans cap10 was constructed successfully.2. Cryptococcus neoformans cells transfected with ps4.1 neo-cap10 are obtained successfully.3.The system of detection of cap10 gene by FQ-PCR was stable and accurate with excellent sensitivity (10⁴ copies/μl),specificity and reproducibility. And it is sufficient enough for the quantitive detection of cryptococcus neoformans cap10 gene.4.The effection of RNA interference on the expression of cryptococcus neoformans cap10 gene is obvious, and it can induce the depression of the ability of escaping the phagocytosis on cryptococcus neoformans.