PHA基因原核表达载体的构建及功能分析
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摘要
为了分析验证phaA、phaB和phaC三个基因的生物学功能，本实验采用PCR技术，从合成PHA的pseudomanas sp．菌株的亚克隆基因组片段中，分离出phaA、phaB和phaC三个基因片段，定向克隆至原核表达载体pBV220上，构建了三个原核生物表达载体pBV-A、pBV-B和pBV-C，通过对表达载体诱导表达，表达蛋白产物的SDS-PAGE分析、生物活性与功能分析，确定了基因phaA、phaB和phaC的生物学功能。结果表明：
1．利用所提取的开放阅读框架的序列设计三对引物，采用PCR技术，从合成PHA的亚克隆片段中分离出phaA、phaB和phaC三个基因片段，经凝胶电泳分析表明，所克隆的三个基因分子量大小与推测的三个开放阅读框架中基因片段大小一致。
2．将phaA、phaB和phaC片段双酶切后，定向克隆至原核表达载体pBV220，构建了三个原核表达载体pBV-A、pBV-B和pBV-C；经酶切分析表明，所克隆的三个基因phaA、phaB和phaC置于表达载体的正确阅读框架下。
3．利用温度诱导外源基因的表达，发现诱导后2小时外源基因开始表达，所表达蛋白的量随着时间的增加而增加，当诱导时间为4小时后外源基因表达的增加量趋于平稳，确定4小时为较为合适的诱导时间。
4．表达载体pBV-A、pBV-B和pBV-C经诱导表达，发现所表达的蛋白均为可溶性蛋白，没有包涵体出现；蛋白经SDS—PAGE分析表明，基因phaA表达的蛋白分子量为42kDa，与β—酮硫裂解酶分子量大小一致；基因phaB表达的蛋白分子量为26kDa，与乙酰乙酰CoA还原酶分子量大小一致；基因phaC表达的蛋白分子量为63kDa，与PHA合成酶分子量大小一致。
5．将phaA、phaB和phaC基因表达的蛋白产物混和后，加入底物乙酰CoA，于230nm—240nm波长下，对反应产物进行扫描，发现在波长235nm处有明显的PHA特异吸收峰；表明混和物中有PHA的产生，所表达的蛋白具有生物学活性和特定功能，证实所克隆的phaA、phaB和phaC就是合成PHA所需的三个基因。
To indetify the function of phaA, phaB and phaC, three genes were amplifed from the subclone of pseudomanas sp. producing PHAs (Poly- 3 -hydroxyalkanoates) by PCR. PCR products were inserted into pBV-220 with double digestion of restriction enduonuclease. The expression vectors of pBV-A pBV-B and pBV-C were constructed by orientaional cloning. Through SDS-PAGE, bioactivity and function analysis of expressed protein, the function of phaA, phaB and phaC was confirmed. The results were as follows:
1. Using primes designed according to the sequence of three extracted ORFs (openig reading frame). phaA, phaB and phaC were amplifed from the subclone of pseudomanas sp. producing PHA by PCR. The Gel Electrophoresis analysis showed that the molecular weights of cloned phaA, phaB and phaC were equal to fragment speculated from three ORFs.
2. phaA, phaB and phaC were inserted to pBV-220 with double digest of restriction enduonuclease. The expression vectors of pBV-A, pBV-B and pBV-C were constructed by orientaional cloning. Indefication of expression vector with restriction enduonuclease digest showed that phaA phaB and phaC were in right ORFs.
3. The analysis of expressed protein in different time showed that the exoteric gene began to express after 2 hour and the quantity of the protein increased with the time increase. But the quantity of the protein increase slowly when the exoteric gene was induced after 4 hour. The result showed that 4 hour was a proper time.
4. The expressed result of pBV-A, pBV-B and pBV-C showed that the expressed protein was soluble and no inclusion body was been found. SDS-PAGE analysis show the molecular weight of protein expressed by phaA was 42kDa which was equal to 3-ketohilase , the molecular weight of protein expressed by phaB was 26kDa which was equal to acetoacetyl-CoA reductase, and the molecular weight of protein expressed by phaC was 63kDa which was equal to PHA synthase.
5. Adding the acetyl -CoA to the protein mixture of phaA, phaB and phaC, the mixture was analysed with the wavelength of 230-240nm. The results showed that the absorption peak for PHA could be found at the wavelength of 235nm. From all facts given above, it could be confirmed that three cloned genes have bioactivity and biological function synthesizing PHA.
1．利用所提取的开放阅读框架的序列设计三对引物，采用PCR技术，从合成PHA的亚克隆片段中分离出phaA、phaB和phaC三个基因片段，经凝胶电泳分析表明，所克隆的三个基因分子量大小与推测的三个开放阅读框架中基因片段大小一致。
2．将phaA、phaB和phaC片段双酶切后，定向克隆至原核表达载体pBV220，构建了三个原核表达载体pBV-A、pBV-B和pBV-C；经酶切分析表明，所克隆的三个基因phaA、phaB和phaC置于表达载体的正确阅读框架下。
3．利用温度诱导外源基因的表达，发现诱导后2小时外源基因开始表达，所表达蛋白的量随着时间的增加而增加，当诱导时间为4小时后外源基因表达的增加量趋于平稳，确定4小时为较为合适的诱导时间。
4．表达载体pBV-A、pBV-B和pBV-C经诱导表达，发现所表达的蛋白均为可溶性蛋白，没有包涵体出现；蛋白经SDS—PAGE分析表明，基因phaA表达的蛋白分子量为42kDa，与β—酮硫裂解酶分子量大小一致；基因phaB表达的蛋白分子量为26kDa，与乙酰乙酰CoA还原酶分子量大小一致；基因phaC表达的蛋白分子量为63kDa，与PHA合成酶分子量大小一致。
5．将phaA、phaB和phaC基因表达的蛋白产物混和后，加入底物乙酰CoA，于230nm—240nm波长下，对反应产物进行扫描，发现在波长235nm处有明显的PHA特异吸收峰；表明混和物中有PHA的产生，所表达的蛋白具有生物学活性和特定功能，证实所克隆的phaA、phaB和phaC就是合成PHA所需的三个基因。
To indetify the function of phaA, phaB and phaC, three genes were amplifed from the subclone of pseudomanas sp. producing PHAs (Poly- 3 -hydroxyalkanoates) by PCR. PCR products were inserted into pBV-220 with double digestion of restriction enduonuclease. The expression vectors of pBV-A pBV-B and pBV-C were constructed by orientaional cloning. Through SDS-PAGE, bioactivity and function analysis of expressed protein, the function of phaA, phaB and phaC was confirmed. The results were as follows:
1. Using primes designed according to the sequence of three extracted ORFs (openig reading frame). phaA, phaB and phaC were amplifed from the subclone of pseudomanas sp. producing PHA by PCR. The Gel Electrophoresis analysis showed that the molecular weights of cloned phaA, phaB and phaC were equal to fragment speculated from three ORFs.
2. phaA, phaB and phaC were inserted to pBV-220 with double digest of restriction enduonuclease. The expression vectors of pBV-A, pBV-B and pBV-C were constructed by orientaional cloning. Indefication of expression vector with restriction enduonuclease digest showed that phaA phaB and phaC were in right ORFs.
3. The analysis of expressed protein in different time showed that the exoteric gene began to express after 2 hour and the quantity of the protein increased with the time increase. But the quantity of the protein increase slowly when the exoteric gene was induced after 4 hour. The result showed that 4 hour was a proper time.
4. The expressed result of pBV-A, pBV-B and pBV-C showed that the expressed protein was soluble and no inclusion body was been found. SDS-PAGE analysis show the molecular weight of protein expressed by phaA was 42kDa which was equal to 3-ketohilase , the molecular weight of protein expressed by phaB was 26kDa which was equal to acetoacetyl-CoA reductase, and the molecular weight of protein expressed by phaC was 63kDa which was equal to PHA synthase.
5. Adding the acetyl -CoA to the protein mixture of phaA, phaB and phaC, the mixture was analysed with the wavelength of 230-240nm. The results showed that the absorption peak for PHA could be found at the wavelength of 235nm. From all facts given above, it could be confirmed that three cloned genes have bioactivity and biological function synthesizing PHA.
引文
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