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目的 :为设计研制安全有效的人脑型疟疫苗提供理论依据。方法 :根据 MAD2 0株裂殖子表面蛋白 1( MSP1)和 FC2 7株裂殖子表面蛋白 2 ( MSP2 )基因编码区高度保守碱基设计并合成两对引物 ,应用多聚酶链反应 ( PCR)技术对 5例脑型疟患者恶性疟原虫云南省勐腊县勐罕分离株 CMH/ YN和云南省盈江县农场 CYJ/ YN分离株基因组 DNA MSP1第 13- 17区基因和MSP2基因进行扩增 ,并将扩增产物分别经 Eco RI和 Kpn I,Bam HI和 Hind III双酶切后 ,分子定向克隆 M13mp18和 M13mp19载体 ,转染大肠杆菌 ( E.coli) TG1,从含 X- gal和 IPTG的 LB平板上 ,将随机筛选得到的单个无色噬菌斑经 E.coli JM10 3扩增 ,用碱裂解法抽提重组子复制型DNA ( RFDNA)后 ,再分别经 Eco RI和 Kpn I,Bam HI和 Hind III双酶切鉴定。结果 :证实重组子为编码脑型疟患者恶性疟原虫 CMH/ YN和 CYJ/ YN分离株 MSP1第 16- 17区基因和 MSP2基因分子克隆 M13载体。结论 :首次报道确证脑型疟患者恶性疟原虫 CMH/ YN和 CYJ/ YN分离株MSP1第 16- 17区基因和 MSP2基因分别与 MAD2 0株 MSP1和 FC2 7株 MSP2相应基因完全一致。这些发现对研究预防人脑型疟疫苗和建立一种新型脑型疟恶性疟原虫检测方法具有重要意义。
Objective: To provide a theoretical basis for designing a safe and effective human brain-based malaria vaccine. METHODS: Two pairs of primers were designed and synthesized according to the highly conserved bases in MAD2 0 and MSP2 gene regions of MAD2 0 gene. Polymerase chain reaction (PCR) 5 cases of cerebral malaria patients with Plasmodium falciparum Mengla County in Yunnan Province Menghan isolate CMH / YN and Yunnan Yingjiang County farm CYJ / YN isolates Genomic DNA MSP1 13-17 gene and MSP2 gene amplification, and The amplified product was double digested with Eco RI and Kpn I, Bam HI and Hind III, respectively, and the M13mp18 and M13mp19 vectors were molecularly cloned and transfected into E. coli TG1. On a plate, a single plaque obtained by random screening was amplified by E. coli JM10 3, and then the recombinant DNA was extracted by alkaline lysis and then by Eco RI and Kpn I, Bam HI And Hind III double digestion identification. Results: The recombinants were confirmed to be the 16-17th gene of MSP1 and the MSP2 gene clone M13 vector of Plasmodium falciparum CMH / YN and CYJ / YN isolates encoding cerebral malaria patients. CONCLUSION: The 16th-17th gene and MSP2 gene of MSP1 of CMH / YN and CYJ / YN isolates of Plasmodium falciparum and Plasmodium falciparum were the first reported to be completely consistent with MSP2 and MSP2 corresponding genes respectively. These findings are of great significance for the study of preventing human cerebral malaria vaccine and establishing a new detection method for Plasmodium falciparum of cerebral malaria.