摘要
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 an attenuated Salmonella TPHK carrying hypoxia inducible factor（HIF）-α and keratinocyte growth factor（KGF） genes, and determine the expression of the two genes in gastrointestinal tissue of rats with hypoxia stress. Methods KGF gene was amplified from plasmid p IRES-KGF by PCR, digested with XbaⅠ and NotⅠ,and inserted into vector p IRES-HIF. The obtained plasmid p IRES-HIF-KGF was transformed to attenuated Salmonella Ty21 a by calcium chloride method. Rats were treated with the constructed recombinant Salmonella strain TPHK by gavage,109 cfu for each daily, for 7 d, and transferred to the hypoxia cabin with mimic environment of plateau at a height of 6 000 m above sea level and further raised for 7 d. The gastrointestinal tissues of rats were collected and determined for the expression levels of HIF-1α and KGF by ELISA. Results Restriction analysis and sequencing proved that recombinant plasmid p IRES-HIF-KGF and recombinant Salmonella TPHK were constructed correctly. The expression levels of HIF-1αand KGF in gastrointestinal tissues of rats increased significantly via gavage with TPHK strain（P〈0. 001）. Conclusion An attenuated Salmonella TPHK strain carrying HIF-1α and KGF genes was successfully constructed, which increased the expression levels of the two genes in gastrointestinal tissue of rats with hypoxia stress.
出处
《中国生物制品学杂志》
CAS
CSCD
2017年第2期137-140,共4页
Chinese Journal of Biologicals
基金
兰州军区兰州总医院重点课题(2013-1015)
关键词
低氧诱导因子-1Α
角质细胞生长因子
减毒沙门菌
大鼠
Hypoxia inducible factor（HIF）-1α
Keratinocyte growth factor（KGF）
Attenuated Salmonella
Rat
