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目的构建携带低氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)及角质细胞生长因子(keratinocyte growth factor,KGF)的减毒沙门菌TPHK,并检测其在低氧应激大鼠胃肠组织中的表达。方法从质粒p IRES-KGF中PCR扩增获得KGF基因片段,经双酶切回收后,与p IRES-HIF质粒进行连接,获得双基因重组质粒p IRES-HIF-KGF,Ca Cl2法转化减毒沙门菌Ty21a,获得重组菌株TPHK。将TPHK菌液灌胃给予大鼠,10~9 cfu/(只·d),饲养7 d后,置模拟海拔6 000 m的高原低氧舱,舱内连续饲养7 d,采集大鼠胃及小肠组织,ELISA法检测HIF-1α及KGF的表达水平。结果重组质粒p IRES-HIF-KGF及重组菌株TPHK经双酶切及测序鉴定证明构建正确。给予TPHK菌液后可明显提高低氧应激大鼠胃及肠组织中HIF-1α及KGF的表达水平(P<0.001)。结论成功构建了携带HIF-1α及KGF的减毒沙门菌TPHK,且其可提高HIF-1α及KGF在低氧应激大鼠胃肠组织中的表达。
Objective To construct attenuated Salmonella typhimurium TPHK carrying hypoxia inducible factor-1α (HIF-1α) and keratinocyte growth factor (KGF), and to detect the expression of TPHK in hypoxic stress rat stomach Expression in intestinal tissue. Methods KGF gene fragment was amplified by PCR from plasmid pIRES-KGF. After double digestion, it was ligated with pIRES-HIF plasmid to obtain pIRES-HIF-KGF. The CaCl 2 gene was transformed and attenuated Salmonella Ty21a, the recombinant strain TPHK was obtained. The TPHK solution was intragastrically administered to rats at 10-9 cfu / (d · d) for 7 d. The plateau hypoxic tank at a simulated altitude of 6 000 m was set up and housed continuously in the tank for 7 days. Small intestine tissue and ELISA were used to detect the expression of HIF-1α and KGF. Results The recombinant plasmid pESES-HIF-KGF and the recombinant strain TPHK were confirmed by double enzyme digestion and sequencing. The TPHK bacteria solution could obviously improve the expression of HIF-1α and KGF in the stomach and intestine of hypoxia stress rats (P <0.001). Conclusion The attenuated Salmonella typhimurium TPHK carrying HIF-1α and KGF was successfully constructed and could increase the expression of HIF-1α and KGF in the gastrointestinal tissues of hypoxic rats.