htrA、clpP基因?qū)ψ儺愭溓蚓L和耐酸性影響
發(fā)布時間：2018-08-13 18:39
【摘要】：目的研究htrA. clpP基因缺失對變異鏈球菌生長及耐酸性影響。 方法利用變異鏈球菌標準株及前期實驗成功構(gòu)建的htrA單基因缺陷株、clpP單基因缺陷株及htrA-clpP雙基因缺陷株,測定4種菌株在正常培養(yǎng)條件下不同時間段的吸光度,采用MTT試驗測定4種菌株在不同時間段的甲躦吸光度變化情況并探究其與平板活菌計數(shù)法所得的活菌量之間關(guān)系；將4種菌株培養(yǎng)至對數(shù)中期,分成兩組,一組作為實驗組進行5小時預酸化處理(pH=5.5),一組作為對照組在普通TPY培養(yǎng)液中進行5小時處理,然后將兩組菌株在致死性酸環(huán)境(pH=3.0)處理3小時,每隔1小時取菌液采用平板活菌計數(shù)法測定活菌數(shù),計算得到生存率,統(tǒng)計學分析。 結(jié)果1.在正常營養(yǎng)條件下,標準株的生長快于htrA、htrA-clpP缺陷株(P0.05),除3-4小時外標準株與clpP缺陷株生長無明顯差異(P0.05),clpP缺陷株生長快于其他兩種缺陷株(P0.05),但htrA缺陷株與htrA-clpP缺陷株生長無明顯差異(P0.05)；2.未經(jīng)預酸化理4種菌株的生存率中,3小時內(nèi),標準株、htrA單基因缺陷株和clpP單基因缺陷株的生存率并無明顯差異(P0.05),而htrA-clpP雙基因缺陷株變化明顯,差異有統(tǒng)計學意義(P0.05),預酸化處理后,與標準株相比,htrA單基因缺陷株、clpP單基因缺陷株及htrA-clpP雙基因缺陷株的生存率都有下降(P0.05)；3種缺陷株相比生存率差異沒有明顯差異(P0.05),預酸化處理的標準株和htrA單基因缺陷株、htrA-clpP雙基因缺陷株生存率均明顯高于未經(jīng)預酸化處理(P0.05),預酸化處理對生存率的影響大于兩種基因缺失(P0.05)。 結(jié)論1.本實驗中htrA、clpP缺失均可抑制變異鏈球菌的生長,并且雙基因缺陷對細菌生長抑制作用與htrA單獨缺失相似,MTT試驗可在一定的菌數(shù)范圍內(nèi)反映標準株與三種缺陷株的活菌數(shù)；2.與變異鏈球菌標準株相比,htrA、clpP基因單獨或同時缺失都使菌株耐酸能力有不同程度的下降。
[Abstract]:Objective to study the effect of htrA. clpP gene deletion on the growth and acid tolerance of Streptococcus mutans. Methods using the standard strain of Streptococcus mutans and the successful construction of htrA single gene deficiency strain and htrA-clpP double gene deficiency strain, the absorbance of four strains were measured under normal culture conditions. MTT test was used to determine the absorbance changes of the four strains in different time periods and to explore the relationship between them and the live bacteria quantity obtained by the method of counting live bacteria on a flat plate, and the four strains were cultured to the middle of logarithmic period and divided into two groups. One group was treated with 5-hour preacidification (pH=5.5) as experimental group, the other group was treated in normal TPY culture medium for 5 hours as control group, then the two groups were treated in lethal acid environment (pH=3.0) for 3 hours. The number of viable bacteria was determined by plate counting method every 1 hour. The survival rate was calculated and analyzed statistically. Result 1. Under normal nutrition condition, the growth of the standard strain was faster than that of the htrAgna htrA-clpP deficiency strain (P0.05). There was no significant difference between the standard strain and the clpP deficient strain except 3-4 hours (P0.05), but there was no significant difference between the htrA deficient strain and the htrA-clpP deficient strain (P0.05). 2. The survival rate of the four strains without preacidification was not significantly different from that of the standard strain within 3 hours (P0.05), but that of the htrA-clpP double gene deficient strain was significantly different from that of the standard strain (P0.05), while the survival rate of the standard strain was not significantly different from that of the htrA-clpP strain (P0.05). The difference was statistically significant (P0.05). Compared with the standard strain, the survival rate of clpP single gene deficient strain and htrA-clpP double gene deficient strain decreased after preacidification (P0.05). There was no significant difference in survival rate among the three defective strains (P0.05). The survival rates of standard strains and htrA single-gene deficient strains were significantly higher than those of non-preacidified strains (P0.05). The effect of preacidification on survival rate was significant. The noise was greater than that of two gene deletions (P0.05). Conclusion 1. In this experiment, the growth of Streptococcus mutans could be inhibited by the deletion of htrAfion clpP, and the inhibitory effect of double gene deficiency on bacteria growth was similar to that of htrA alone. MTT test could reflect the number of live bacteria of standard strain and three defective strains in a certain range. Compared with the standard strain of Streptococcus mutans, the deletion of htrAgna clpP gene alone or at the same time decreased the acid-tolerance ability of the strain to varying degrees.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
本文編號：2181841
[Abstract]:Objective to study the effect of htrA. clpP gene deletion on the growth and acid tolerance of Streptococcus mutans. Methods using the standard strain of Streptococcus mutans and the successful construction of htrA single gene deficiency strain and htrA-clpP double gene deficiency strain, the absorbance of four strains were measured under normal culture conditions. MTT test was used to determine the absorbance changes of the four strains in different time periods and to explore the relationship between them and the live bacteria quantity obtained by the method of counting live bacteria on a flat plate, and the four strains were cultured to the middle of logarithmic period and divided into two groups. One group was treated with 5-hour preacidification (pH=5.5) as experimental group, the other group was treated in normal TPY culture medium for 5 hours as control group, then the two groups were treated in lethal acid environment (pH=3.0) for 3 hours. The number of viable bacteria was determined by plate counting method every 1 hour. The survival rate was calculated and analyzed statistically. Result 1. Under normal nutrition condition, the growth of the standard strain was faster than that of the htrAgna htrA-clpP deficiency strain (P0.05). There was no significant difference between the standard strain and the clpP deficient strain except 3-4 hours (P0.05), but there was no significant difference between the htrA deficient strain and the htrA-clpP deficient strain (P0.05). 2. The survival rate of the four strains without preacidification was not significantly different from that of the standard strain within 3 hours (P0.05), but that of the htrA-clpP double gene deficient strain was significantly different from that of the standard strain (P0.05), while the survival rate of the standard strain was not significantly different from that of the htrA-clpP strain (P0.05). The difference was statistically significant (P0.05). Compared with the standard strain, the survival rate of clpP single gene deficient strain and htrA-clpP double gene deficient strain decreased after preacidification (P0.05). There was no significant difference in survival rate among the three defective strains (P0.05). The survival rates of standard strains and htrA single-gene deficient strains were significantly higher than those of non-preacidified strains (P0.05). The effect of preacidification on survival rate was significant. The noise was greater than that of two gene deletions (P0.05). Conclusion 1. In this experiment, the growth of Streptococcus mutans could be inhibited by the deletion of htrAfion clpP, and the inhibitory effect of double gene deficiency on bacteria growth was similar to that of htrA alone. MTT test could reflect the number of live bacteria of standard strain and three defective strains in a certain range. Compared with the standard strain of Streptococcus mutans, the deletion of htrAgna clpP gene alone or at the same time decreased the acid-tolerance ability of the strain to varying degrees.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378
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，本文編號：2181841
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