弓形虫P24基因敲除转染质粒pGB/P5-P3的构建

Construction of the transfecting plasmid pGB/P5-P3 with the P24 gene of Toxoplasma gondii knocked out

目的构建弓形虫P24(TgP24)基因敲除转染质粒pGB/P5P3(GRA2/BleP245’UTRP243,UTR),为TgP24基因敲除奠定基础。方法根据TgP24基因序列,设计并合成两对特异引物(P1toP4),采用PCR技术特异扩增TgP24基因的5,端非翻译区2.5kb片段(P245’UTR)和3’端非翻译区2.89kb片段(P243’UTR),将其分别亚克隆入pCR2.1TOPOTA载体,构建质粒P245’UTR/TA和P243’UTR/TA;重组质粒P245’UTR/TA经KpnⅠ和BglⅡ双酶切后,再将纯化的P245’UTR片段亚克隆入转染质粒GRA2/Ble的KpnⅠ和BglⅡ位点,构建重组质粒pGBP5(GRA2/BleP245’UTR);重组质粒P243’UTR/TA经BamHⅠ和NotⅠ双酶切后,纯化P243’UTR片段,再将其定向克隆到重组质粒pGBP5的BamHⅠ和NotⅠ位点,从而构建弓形虫TgP24基因敲除转染质粒pGB/P5P3。重组质粒经DNA序列测定证实目的片段插入正确。结果经过PCR筛选、限制性酶切及DNA测序鉴定,证实P245’UTR和P243’UTR两片段正确插入质粒GRA2/Ble的KpnⅠ和BglⅡ及BamHⅠ和NotⅠ位点,位于药物选择ble基因的上,下游。结论成功构建弓形虫P24基因敲除转染质粒pGB/P5P3。

The aim of this study is to construct the transfecting plasmid with the P24 of Toxoplasma gondii (TgP24) knocked out for the development of the foundation forTgP24 gene knockout.According to the genomic sequences of TgP24, 4 oligonucleotides primers (P1 P4) were designed and synthesized to amplify the 5' end 2.5 kb fragment in un translated region (UTR) and the 3' end 2.89 kb fragment of UTR in Tg24. The PCR products were subcloned into pCR2.1 TOPO TA plasmid to generate the recombinant plasmid p24 5' UTR/TA and P24 3' UTR/TA respectively. The purified fragment of P24 5' UTR by the digestion of P24 5' UTR/TA with Kpn1 and Bgl II was inserted into the Kpn I and Bgl II sites of plasmid GRA2/Ble to genrate plasmind GRA2/Ble P24 5' UTR (designated as pGB P5); and the purified P24 3' UTR fragment by the digestion of plasmid P24 3' UTR/TA with BamH I and Not I was inserted into the BamH I and Not I sites of plasmid pGB P5 to generate the targeting plasmid pGB/P5 P3 (pGB P5/P24 3' UTR). Sequence analysis was performed to confirm that no PCR mutation was introduced. Experimental results showed that the targeting plasmid PGB/P5 P3 was constructed by inserting 2.5 and 2.89 kb DNA fragments corresponding to the 5'end and the 3' end UTR of the TgP24 gene into the plasmid GRA2/Ble.The fragment P24 5'UTR was inserted into the poly linker site Kpn I/Bgl II upstream of the bleomycin resistance gene (ble), and the P24 3'UTR frament inserted into BamH I/Not I site down stream of the same gene. Meanwhile,the orientation of the cloned gene fragments was determined by restriction and sequencing analysis.It is concluded that the transfecting plasmid pGB/P5 P3 is successfully constructed in the present study.