弓形虫表面抗原P30基因分枝杆菌-大肠杆菌穿梭表达重组质粒的构建及序列测定

目的　构建编码弓形虫RH株表面抗原P30基因的分枝杆菌 -大肠杆菌穿梭表达重组质粒并进行其序列测定。方法　弓形虫RH株腹腔接种小鼠 ,收集腹水 ,酚 /氯仿法抽提基因组DNA ;根据基因库P30基因序列设计合成一对引物 ,采用PCR法扩增编码P30的基因片段 ,经低熔点琼脂糖法回收并纯化 ;将P30基因定向克隆到分枝杆菌 -大肠杆菌穿梭表达质粒 ,转化大肠杆菌DH5dα,在卡那霉素阳性LB培养基平板筛选阳性重组子 ,并经双酶切及PCR鉴定 ;最后对重组子进行序列测定。结果　PCR所扩增的P30基因片段为 10 37bp ,阳性重组质粒pBCG -P30经XbaI+KpnI双酶切 ,获得包含P30和热休克蛋白 (hsp70 )启动子的复合基因片段 ,此片段的大小为 1170bp ,与预期的理论值相符合。序列测定分析进一步表明所克隆的基因为编码P30抗原的基因片段。结论　成功构建编码弓形虫表面抗原P30基因大肠杆菌 -分枝杆菌穿梭质粒pBCG -P30。

CONSTRUCTION AND SEQUENCE DETERMINATION OF E. COLI-MYCOBACTERIA RECOMBINANT SHUTTLE PLASMID OF P30 GENE FRAGMENT OF TOXOPLASMA GONDII

Aim To construct a E.coli-Mycobacteria recombinant shuttle plasmid containing P30 gene of Toxoplasma gondii. Methods Injecting mice with RH strain tachyzoites then harvesting infected ascites of mice ,genomic DNA of Toxoplasma gondii was extracted with phenol/chloroform. A pair of primers were designed according to the sequence of P30 gene, a fragment of gene coding P30 antigen was amplified by means of polymerase chain reaction(PCR).The purified PCR products and pBCG5.6 plasmid were digested by BamHI+KpnI and ligated by T4 ligase, the recombinant shuttle expression plasmid, pBCG-P30, was transferred into E.coli DH5α,positive clones were screened and identified by endonulease digestion and PCR technique. Sequence determination analysis was performed to identify the cloned gene. Results The P30 gene fragment with about 1037 base pairs were specifically amplified by using PCR technique. The positive recombinant plasmid pBCG-P30 was screened and digested by XbaI+KpnI ,a compound gene fragment of including P30 and hsp70 promoter gene was obtained , the size of compound fragment is 1170 bp and in accordance with the expected one. Sequence determination analysis showed that the cloned gene was gene fragment coding P30 antigen. Conclusion The E.coli-Mycobacteria shuttle expression plasmids containing P30 gene from Toxoplasma gondii were constructed successfully.