中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2015年
3期
259-265
,共7页
张彦新%曾雪芳%刘芳%蒋云生
張彥新%曾雪芳%劉芳%蔣雲生
장언신%증설방%류방%장운생
乳酸乳球菌%尿酸氧化酶%质粒%NICE表达系统
乳痠乳毬菌%尿痠氧化酶%質粒%NICE錶達繫統
유산유구균%뇨산양화매%질립%NICE표체계통
Lactococcus lactis%Urate oxidase%Plasmid%NICE expression system
目的 将尿酸氧化酶基因克隆到乳酸乳球菌(Llactis)NZ9000中,使之能启动nisA放大系统增加尿酸氧化酶活性,构建一株高效分解尿酸的基因工程菌.方法 根据GenBank上已知的产朊假丝酵母菌尿酸氧化酶基因序列(Uricase,E12709)设计引物,PCR扩增尿酸氧化酶基因片段,将其克隆入质粒PNZ8048、PMG36e,构建重组质粒PNZ8048-U、PMG36e-U,重组质粒电转化L.lactis NZ9000构建基因工程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(SDS-PAGE)鉴定重组菌体裂解液中的尿酸氧化酶,测定尿酸氧化酶的活性和在高尿酸患者血清中降解尿酸的能力.结果 构建的重组质粒PNZ8048-U、PMG36e-U经酶切和测序显示,尿酸氧化酶基因片段长度为0.9 kb,基因片段序列与GenBank上尿酸氧化酶基因序列完全一致.构建重组基因工二程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,均表达相对分子质量约为34 000的重组蛋白,与从尿酸氧化酶基因序列推测的303个氨基酸的理论分子量相符.体外测定菌酶液活性,L.lactis NZ9000-PNZ8048-U组酶活性为(1.92±0.14)u/ml,产朊假丝酵母菌组酶活性为(0.55±0.05)u/ml,L.lactis NZ9000-PMG36e-U组酶活性为(0.29±0.06) u/ml.高尿酸血症患者血清培养结果显示加入生理盐水的对照组尿酸值(620.0±58.7) μmol/L,L.lactis NZ9000-PNZ8048-U组(321.0±46.2) μmol/L,L.lactis NZ9000-PMG36e-U组(568.0±47.3)μmol/L,产朊假丝酵母菌组(406.0±42.4)μmol/L,其他3组与对照组比较,差异均有统计学意义(均P＜0.05).结论 成功构建基因工程菌L.lactis NZ9000-PNZ8048-U,重组菌酶活性较基因来源菌产朊假丝酵母菌高,并可高效分解高尿酸患者血清中的尿酸.
目的 將尿痠氧化酶基因剋隆到乳痠乳毬菌(Llactis)NZ9000中,使之能啟動nisA放大繫統增加尿痠氧化酶活性,構建一株高效分解尿痠的基因工程菌.方法 根據GenBank上已知的產朊假絲酵母菌尿痠氧化酶基因序列(Uricase,E12709)設計引物,PCR擴增尿痠氧化酶基因片段,將其剋隆入質粒PNZ8048、PMG36e,構建重組質粒PNZ8048-U、PMG36e-U,重組質粒電轉化L.lactis NZ9000構建基因工程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,經十二烷基硫痠鈉-聚丙烯酰氨凝膠電泳(SDS-PAGE)鑒定重組菌體裂解液中的尿痠氧化酶,測定尿痠氧化酶的活性和在高尿痠患者血清中降解尿痠的能力.結果 構建的重組質粒PNZ8048-U、PMG36e-U經酶切和測序顯示,尿痠氧化酶基因片段長度為0.9 kb,基因片段序列與GenBank上尿痠氧化酶基因序列完全一緻.構建重組基因工二程菌L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,均錶達相對分子質量約為34 000的重組蛋白,與從尿痠氧化酶基因序列推測的303箇氨基痠的理論分子量相符.體外測定菌酶液活性,L.lactis NZ9000-PNZ8048-U組酶活性為(1.92±0.14)u/ml,產朊假絲酵母菌組酶活性為(0.55±0.05)u/ml,L.lactis NZ9000-PMG36e-U組酶活性為(0.29±0.06) u/ml.高尿痠血癥患者血清培養結果顯示加入生理鹽水的對照組尿痠值(620.0±58.7) μmol/L,L.lactis NZ9000-PNZ8048-U組(321.0±46.2) μmol/L,L.lactis NZ9000-PMG36e-U組(568.0±47.3)μmol/L,產朊假絲酵母菌組(406.0±42.4)μmol/L,其他3組與對照組比較,差異均有統計學意義(均P＜0.05).結論 成功構建基因工程菌L.lactis NZ9000-PNZ8048-U,重組菌酶活性較基因來源菌產朊假絲酵母菌高,併可高效分解高尿痠患者血清中的尿痠.
목적 장뇨산양화매기인극륭도유산유구균(Llactis)NZ9000중,사지능계동nisA방대계통증가뇨산양화매활성,구건일주고효분해뇨산적기인공정균.방법 근거GenBank상이지적산원가사효모균뇨산양화매기인서렬(Uricase,E12709)설계인물,PCR확증뇨산양화매기인편단,장기극륭입질립PNZ8048、PMG36e,구건중조질립PNZ8048-U、PMG36e-U,중조질립전전화L.lactis NZ9000구건기인공정균L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,경십이완기류산납-취병희선안응효전영(SDS-PAGE)감정중조균체렬해액중적뇨산양화매,측정뇨산양화매적활성화재고뇨산환자혈청중강해뇨산적능력.결과 구건적중조질립PNZ8048-U、PMG36e-U경매절화측서현시,뇨산양화매기인편단장도위0.9 kb,기인편단서렬여GenBank상뇨산양화매기인서렬완전일치.구건중조기인공이정균L.lactis NZ9000-PNZ8048-U、L.lactis NZ9000-PMG36e-U,균표체상대분자질량약위34 000적중조단백,여종뇨산양화매기인서렬추측적303개안기산적이론분자량상부.체외측정균매액활성,L.lactis NZ9000-PNZ8048-U조매활성위(1.92±0.14)u/ml,산원가사효모균조매활성위(0.55±0.05)u/ml,L.lactis NZ9000-PMG36e-U조매활성위(0.29±0.06) u/ml.고뇨산혈증환자혈청배양결과현시가입생리염수적대조조뇨산치(620.0±58.7) μmol/L,L.lactis NZ9000-PNZ8048-U조(321.0±46.2) μmol/L,L.lactis NZ9000-PMG36e-U조(568.0±47.3)μmol/L,산원가사효모균조(406.0±42.4)μmol/L,기타3조여대조조비교,차이균유통계학의의(균P＜0.05).결론 성공구건기인공정균L.lactis NZ9000-PNZ8048-U,중조균매활성교기인래원균산원가사효모균고,병가고효분해고뇨산환자혈청중적뇨산.
Objective To construct a bacterial strain with genectically-engineered high-efficiency in uric acid degradation,through cloning the urate oxidase gene into Lactococcus lactis NZ9000 so that it can promote nisA amplification system to increase urate oxidase activity.Methods According to the urate oxidase gene sequence (Uricase,E12709) of Candida utilis retrieved in GenBank,PCR was used to amplify the Uricase sequence with predesigned primers.The amplified gene fragment was cloned into plasmids PNZ8048 and PMG36e to form the the recombinant plasmids PNZ8048-U and PMG36e-U,which were then transformed into L.lactis NZ9000 and finally we obtained two genetically-engineered strains,L.lactis NZ9000-PNZ8048-U and L.lactis NZ9000-PMG36e-U.SDS-PAGE was used to detect the urate oxidase and its activity in the lysate of recombinant bacteria.The uric acid degradation by the strains in serum from patients with hyperuricemia was measured.Results The recombinant plasmids PNZ8048-U and PMG36e-U were identified by enzyme digestion and sequencing,showing a 0.9 kb urate oxidase gene fragment identical to the GenBank data.Both of the recombinant strains,L.lactis NZ9000-PNZ8048-U and L.lactis NZ9000-PMG36e-U,expressed a recombinant protein (relative molecular mass 34 000)which was consistent with the theoretical molecular weight of the putative 303 amino acids of urate oxidase gene sequence.In-vitro measurement showed that the urate oxidase activity of L.Lactis NZ9000-PNZ8048-U was (1.92±0.14) u/ml,compared with (0.55±0.05) u/ml for Candida utilis and (0.29±0.06) u/ml for L.Lactis NZ9000-PMG36e-U.The serum uric acid levels of patients with hyperuricemia were (620.0 ±58.7) μmol/L in samples added with normal saline,(321.0 ± 46.2) μmol/L in samples added with L.lactis NZ9000-PNZ8048-U,(568.0 ± 47.3) μmol/L in samples added with L.lactis NZ9000-PMG36e-U,and (406.0 ±42.4) μmol/L in samples added with Candida utilis.There was a statistical difference in the serum uric acid level between the samples added with bacteria and those in the negative control group (all P＜0.05).Conclusion A genetically engineered strain L.lactis NZ9000-PNZ8048-U was successfully constructed in this study.Compared with the gene-source,Candida utilis,the recombinant bacterial strain showed higher activity of urate oxidase and higher efficiency in degradation of serum uric acid from hyperuricemic patients.
