CAJ | 학술논문

目的寻找休眠结核分枝杆菌复苏的关键基因,探讨结核分枝杆菌复苏的机制.方法取改良7H9培养基培养20 d的结核分枝杆菌标准株H37Rv作为对数生长期的活跃结核分枝杆菌,提取其RNA.将一部分该菌株在37℃密封无氧培养45 d左右,加入亚甲蓝作为无氧标志,即获得休眠菌.向休眠菌中加入复苏促进因子,37℃有氧培养3 d,提取休眠复苏期结核分枝杆菌的基因组RNA.用DNA酶处理RNA并回收RNA,用mRNA纯化试剂盒纯化RNA.利用抑制性消减杂交(Suppression subtractive hybridization,SSH)技术分析休眠复苏期和活跃期结核分枝杆菌的基因组mRNA的表达差异,并通过基因克隆、测序和序列分析,寻找差异表达基因.实时荧光定量PCR鉴定差异表达基因.结果以活跃期结核分枝杆菌cDNA为检测子的正相杂交和以休眠复苏期结核分枝杆菌cDNA为检测子的反相杂交各自高表达或特异性表达的片段都得到了选择性扩增,杂交产物经连接pTA2载体、转化至大肠杆菌DH5?和蓝白斑筛选,正相杂交获得78个阳性菌落,反相杂交获得46个阳性菌落.阳性单个菌落经液体LB培养基培养,以菌液为模板,T7、M13为引物,扩增插入片段,能扩增到明显的条带,且＞350 bp的条带为阳性.正相杂交得到66个阳性扩增带,反相杂交得到39个阳性扩增带.这105个阳性扩增带的菌液经测序分析,则正相有30个特异性序列,反相有21个特异性序列.将这51个序列通过Genbank检索,因有不同序列对应同一基因,最后正相获得了20个活跃结核分枝杆菌特异性表达或高表达的功能基因,反相获得了7个休眠复苏期结核分枝杆菌特异性表达或高表达的功能基因,根据其功能不同分为8大类.经实时荧光定量PCR分析,7个休眠复苏期结核分枝杆菌特异性表达或高表达功能基因的表达量均为活跃结核分枝杆菌的该基因表达量的4倍以上.结论利用SSH技术可以筛选到休眠复苏期结核分枝杆菌和活跃期结核分枝杆菌的差异表达基因,为寻找休眠结核分枝杆菌复苏的关键基因奠定了基础. 目的尋找休眠結覈分枝桿菌複囌的關鍵基因,探討結覈分枝桿菌複囌的機製.方法取改良7H9培養基培養20 d的結覈分枝桿菌標準株H37Rv作為對數生長期的活躍結覈分枝桿菌,提取其RNA.將一部分該菌株在37℃密封無氧培養45 d左右,加入亞甲藍作為無氧標誌,即穫得休眠菌.嚮休眠菌中加入複囌促進因子,37℃有氧培養3 d,提取休眠複囌期結覈分枝桿菌的基因組RNA.用DNA酶處理RNA併迴收RNA,用mRNA純化試劑盒純化RNA.利用抑製性消減雜交(Suppression subtractive hybridization,SSH)技術分析休眠複囌期和活躍期結覈分枝桿菌的基因組mRNA的錶達差異,併通過基因剋隆、測序和序列分析,尋找差異錶達基因.實時熒光定量PCR鑒定差異錶達基因.結果以活躍期結覈分枝桿菌cDNA為檢測子的正相雜交和以休眠複囌期結覈分枝桿菌cDNA為檢測子的反相雜交各自高錶達或特異性錶達的片段都得到瞭選擇性擴增,雜交產物經連接pTA2載體、轉化至大腸桿菌DH5?和藍白斑篩選,正相雜交穫得78箇暘性菌落,反相雜交穫得46箇暘性菌落.暘性單箇菌落經液體LB培養基培養,以菌液為模闆,T7、M13為引物,擴增插入片段,能擴增到明顯的條帶,且＞350 bp的條帶為暘性.正相雜交得到66箇暘性擴增帶,反相雜交得到39箇暘性擴增帶.這105箇暘性擴增帶的菌液經測序分析,則正相有30箇特異性序列,反相有21箇特異性序列.將這51箇序列通過Genbank檢索,因有不同序列對應同一基因,最後正相穫得瞭20箇活躍結覈分枝桿菌特異性錶達或高錶達的功能基因,反相穫得瞭7箇休眠複囌期結覈分枝桿菌特異性錶達或高錶達的功能基因,根據其功能不同分為8大類.經實時熒光定量PCR分析,7箇休眠複囌期結覈分枝桿菌特異性錶達或高錶達功能基因的錶達量均為活躍結覈分枝桿菌的該基因錶達量的4倍以上.結論利用SSH技術可以篩選到休眠複囌期結覈分枝桿菌和活躍期結覈分枝桿菌的差異錶達基因,為尋找休眠結覈分枝桿菌複囌的關鍵基因奠定瞭基礎.
목적 심조휴면결핵분지간균복소적관건기인,탐토결핵분지간균복소적궤제.방법 취개량7H9배양기배양20 d적결핵분지간균표준주H37Rv작위대수생장기적활약결핵분지간균,제취기RNA.장일부분해균주재37℃밀봉무양배양45 d좌우,가입아갑람작위무양표지,즉획득휴면균.향휴면균중가입복소촉진인자,37℃유양배양3 d,제취휴면복소기결핵분지간균적기인조RNA.용DNA매처리RNA병회수RNA,용mRNA순화시제합순화RNA.이용억제성소감잡교(Suppression subtractive hybridization,SSH)기술분석휴면복소기화활약기결핵분지간균적기인조mRNA적표체차이,병통과기인극륭、측서화서렬분석,심조차이표체기인.실시형광정량PCR감정차이표체기인.결과 이활약기결핵분지간균cDNA위검측자적정상잡교화이휴면복소기결핵분지간균cDNA위검측자적반상잡교각자고표체혹특이성표체적편단도득도료선택성확증,잡교산물경련접pTA2재체、전화지대장간균DH5?화람백반사선,정상잡교획득78개양성균락,반상잡교획득46개양성균락.양성단개균락경액체LB배양기배양,이균액위모판,T7、M13위인물,확증삽입편단,능확증도명현적조대,차＞350 bp적조대위양성.정상잡교득도66개양성확증대,반상잡교득도39개양성확증대.저105개양성확증대적균액경측서분석,칙정상유30개특이성서렬,반상유21개특이성서렬.장저51개서렬통과Genbank검색,인유불동서렬대응동일기인,최후정상획득료20개활약결핵분지간균특이성표체혹고표체적공능기인,반상획득료7개휴면복소기결핵분지간균특이성표체혹고표체적공능기인,근거기공능불동분위8대류.경실시형광정량PCR분석,7개휴면복소기결핵분지간균특이성표체혹고표체공능기인적표체량균위활약결핵분지간균적해기인표체량적4배이상.결론 이용SSH기술가이사선도휴면복소기결핵분지간균화활약기결핵분지간균적차이표체기인,위심조휴면결핵분지간균복소적관건기인전정료기출.
Objective To screen key genes of dormant M. tuberculosis for resuscitation.Methods M.tuberculosis H37 Rv strain cultured for 20 days in 7H9 liquid medium was used as active bacteria.Dormant bacteria were obtained by cultivating active bacteria hermetically at 37 ℃ using methylene blue as the indicator of oxygen free until the blue medium became colorless.Then resuscitation promoting factors were added to the culture and the bacteria were cultivated for 3 days to be resuscitated.RNA was extracted from active bacteria and resuscitating bacteria.disposed of DNA in RNA with DNase I,and mRNA was purified and then were hybridized using suppression subtractive hybridization (SSH) technique.Differentially expressed genes between resuscitating M.tuberculosis and active M.tuberculosis were identified by PCR,cloning,and sequence alignment.Identification of the differentially expressed genes was performed by real-time quantitative PCR.Results High or specifically expressed genes as tester had been obtained by SSH in correctitude reaction (active M.tuberculosis as tester) and reverse reaction(dormant M.tuberculosis as tester).These genes were cloned into plasmid PGEM-T Easy.and 78 positive bacteria in correctitude reaction and 46 positive bacteria in reverse reaction were obtained.The positive bacteria were amplified by PCR with T7 and M13 primer.and 66 positive bacteria (＞350 bp) in correctitude reaction and 39 positive bacteria (＞350 bp) in reverse reaction were obtained.After sequencing,30 positive sequences in correctitude reaction and 21 positive sequences in reverse reaction were obtained.Twenty and 7 high or specifically expressed genes were finally identified in active and resuscitating M.tuberculosis respectively by seaiching in Genbank.These genes were classified into 8 categories.Real-time quantitative PCR demonstrated that the quantity of 7 hish or specifically expressed genes in resuscitating bacteria was more than 4 times that in active bacteria.Conclusion Differentially expressed genes between resuscitating and active M.tuberculosis were identified using SSH technique and the results may help exploring key genes and mechanisms of dormant M.tuberculosis for resuscitation.