贝类中诺如病毒的风险评估及与组织血型抗原相关性
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摘要
诺如病毒（Noroviruses，NoVs）是引起世界范围内非细菌性急性肠胃炎的主要病原体，贝类是其最主要的传播载体。人们通常由于食用了生的或者加热不彻底的海产贝类而感染NoVs，并引起急性肠胃炎。我国是世界水产大国，贝类年产量1000多万吨，在我国国民经济中占有较重要的地位，NoVs作为贝类中常见的食源性致病微生物是质量安全一个不容忽视的问题。然而，NoVs尚不可体外培养，贝类中因NoVs含量少、存在PCR抑制剂多等原因给检测带来困难，我国尚未建立贝类中NoVs检测的国家标准；研究表明组织血型抗原是人体感染NoVs的受体，牡蛎中也存在类似的受体，但牡蛎中该受体与NoVs的相关性及其对牡蛎富集NoVs的影响尚不清楚。因此建立贝类中NoVs的富集检测方法，调查我国主要沿海地区贝类中NoVs的分布情况以及开展贝类中NoVs的风险评估；研究牡蛎中NoVs与组织血型抗原相关性，探索牡蛎等贝类富集NoVs的机理，对于保护消费者健康，保障我国贝类产业可持续健康发展具有重要的意义。
本文以贝类中NoVs为研究对象，优化建立了海产贝类中GI和GII型NoVs的富集方法，调查了黄渤海区7个城市零售海产贝类中NoVs的污染状况，在上述研究的基础上，结合CAC的食品安全风险分析理论，开展贝类中NoVs的风险评估研究；针对NoVs受体，建立了太平洋牡蛎中A型组织血型抗原的ELISA检测方法，并调查了威海、青岛、日照3个城市太平洋牡蛎样品中A型组织血型抗原随季节的变化规律；利用RACE技术得到了一个太平洋牡蛎组织血型抗原合成相关基因FUT3的cDNA全长，并进行了原核表达；利用牡蛎全基因组序列，初步探索了温度对牡蛎组织学型抗原合成关键基因FUT2的表达调控。具体研究结果如下：
1．贝类中NoVs富集方法的建立和优化
通过人工污染实验，向牡蛎和菲律宾蛤仔的消化道匀浆物中分别添加不同浓度的GI.3和GII.4型NoVs，利用4种贝类中NoVs富集方法分别回收病毒，用实验室前期建立的real-time RT-PCR方法进行检测。以4种方法从牡蛎和菲律宾蛤仔中富集GI.3和GII.4型NoVs的富集效率评价4种方法的优劣，同时分析影响NoVs富集的主要因素。结果显示方法B（Proteinase K-PEG8000）操作简单，耗时较短，且稳定性和灵敏性均优于其他方法，其从牡蛎和菲律宾蛤仔中富集GI.3型NoVs的富集效率分别为9.3%和9.6%，对GII.4型NoV的富集效率分别为13.1%和12.3%，比其他三种方法的富集效率高（p<0.05）。研究结果还发现贝类品种及贝类中NoVs的含量是影响贝类中NoVs富集效率的主要因素。
2．黄渤海贝类中NoVs污染状况调查
从2009年12月至2011年11月，采集了大连、莱州、烟台、威海、青岛、日照、连云港7个黄渤海区沿海城市6种零售贝类样品840个。调查结果显示NoVs在6种贝类中均有检出，平均检出率为13.33%(112/840)。不同贝类中NoVs的检出率为：牡蛎19.35%，紫贻贝16.67%，扇贝5.70%，缢蛏8.82%，毛蚶13.74%，菲律宾蛤仔16.44%。贝类中NoVs的季节分布结果显示，夏季贝类中NoVs检出率最高，为16.44%，冬季和秋季次之，春季最低。此外，研究发现不同季节NoVs检出率存在明显的品种特异性差异。春季和冬季牡蛎样品中NoVs的检出率最高，夏季毛蚶样品中NoVs的检出率最高，而冬季菲律宾蛤仔中NoVs的检出率最高。在冬季采集的33个扇贝样品中，NoVs无检出。
定量结果显示，112个阳性贝类样品中，96个样品中NoVs含量<103基因拷贝。运用spss17.0软件对不同贝类品种和不同季节贝类中NoV污染水平进行分析，非参检验（Kruskal-Wallis H tests）结果显示不同贝类品种和不同季节贝类中NoV污染水平均无明显差异(p>0.05)。112个阳性样品中，普通RT-PCR复检中仅96个样品琼脂糖凝胶电泳显示目的条带。将这96个样品测序结果输入Norovirus GenotypingTool Version1.0软件后，结果显示94个NoV属于GII.12变异株，2个属于GI.3。在冬季采集的菲律宾蛤仔和毛蚶中检测到GI.3型NoV。
3．贝类中NoVs的风险评估
本研究针对上述情况，利用前期调查研究获得的贝类中NoVs污染状况数据和相关膳食调查数据，对我国海产贝类中NoVs进行了定量风险评估，研究结果发现我国沿海城市居民食用贝类引发肠胃炎的平均概率为4.08E-7，预计每年0-4岁、5-64岁和65岁以上人口的发病数分别为119、9078和339人。本次评估只考虑贝类中NoVs污染率和污染水平以及贝类摄入量对风险的影响，存在一定的不确定性。国内外有关贝类中NoVs风险评估的研究较少，本次评估可以为贝类或其他食品中NoVs的风险评估提供一定的参考，相关调查资料可以为贝类食用安全提供指导意义。
4．牡蛎组织血型抗原的检测
在实验室前期研究的基础上改进前处理方法，建立了太平洋牡蛎中A型组织血型抗原的ELISA检测方法。运用建立的ELISA方法和第二章建立的贝类中NoVs的富集方法对2010年从威海、青岛、日照3个城市采集的36份太平洋牡蛎进行了A型组织血型抗原和NoVs的检测，结果表明三个城市太平洋牡蛎中A型组织血型抗原随季节变化呈现波动性变化，在全年不同月份出现峰值；36份牡蛎样品检出NoVs阳性6份，检出率为16.7%，且检出时间出现在A型组织血型抗原的高峰值处，推测贝类受NoVs污染可能与组织血型抗原表达相关。
5.牡蛎组织血型抗原相关基因的克隆及原核表达
参照人体组织血型抗原合成途径，选择FUT2基因作为牡蛎组织血型抗原合成关键基因。由于当时牡蛎全基因组序列尚未公布，通过Clustal X与Mega等分析软件，分析人、猩猩、小鼠、家犬等物种的FUT2基因氨基酸序列，推测牡蛎FUT2的保守区，根据保守序列设计引物。利用PCR方法扩增牡蛎组织血型抗原合成相关基因得到预期片段488bp的扩增产物，通过牡蛎“FUT2”扩增片段与人、小鼠、猩猩等物种的序列比对分析，可以初步证实牡蛎证实在牡蛎基因组中存在FUT2基因的保守片段。根据PCR方法得到的牡蛎“FUT2”的部分序列，设计引物，利用RACE扩增技术对牡蛎基因组进行扩增，克隆测序得到牡蛎“FUT2”基因3’和5’端序列，序列拼接后得到1086bp的cDNA全长基因。2012年9月，太平洋牡蛎全基因组序列公布，通过比对发现得到的牡蛎“FUT2”蛋白序列与FUT3基因同源性为98%，而与牡蛎FUT2基因的同源性为36%，因此证实所得序列为太平洋牡蛎FUT3基因。在此基础上，通过构建表达载体，成功诱导出FUT3的蛋白，大小为41.5kDa，为后续FUT3基因的功能验证打下基础。
6．温度对牡蛎组织血型抗原的表达调控
以β-actin作为内参基因，建立太平洋牡蛎中FUT2基因的荧光定量PCR检测方法。用高温（28℃）和低温（4℃）处理太平洋牡蛎，分别于48h和72h取样，研究牡蛎外套膜、闭壳肌、鳃和内脏4个组织中7组FUT2基因的表达情况。荧光定量PCR研究结果表明温度对FUT2部分基因的表达存在一定的影响。高温和低温条件，FUT2部分基因存在表达上调的现象。7组基因中，高温胁迫时FUT2.1和FUT2.3基因在牡蛎4个不同组织中表达上调；低温胁迫时，FUT2.4在外套膜和鳃组织中表达上调。
Noroviruses (NoVs) are commonly occurring pathogens that causegastroenteritis. NoVs can be isolated from water, vegetable, fruit andseafood samples, especially in shellfish samples frequently. It usually causesacute gastroenteritis associated with the consumption of raw or undercookedseafood. On rare occasion, infection can lead to severe dehydration and deathmay also occur subsequently. The annual production of shellfish was nearly upto10,000,000tons in China. For shellfish is delicious, the coastal residents arefond of it. Foodborne outbreaks caused by NoVs associated with theconsumption of shellfish were constantly reported in the coastal area of China.More attention should be paid to food safety issuses arised by NoVs.
To investigate the prevalence of NoVs in shellfish, the quantitative detectionmethods were optimized. The level of NoVs in shellfish of seven cities aroundYellow Sea and Bohai Sea in China was investigated by real-time RT-PCR andthen the quantitative assessment was carried out. The recent studies showedthat NoVs recognize histo-blood group antigens (HBGAs) as receptors inoyster.To elucidate the mechanism of NoVs accumulated in oysters, firstly, the ELISA and PCR method was optimized to detect A-type HBGAs and keygenes of HBGAs in oyster. Second, total length cDNA of one key genes FUT3was got and the protein was expressed.Temperature effect on expression levelof FUT2was also studied.The main contents were as follows:
1. The detection methods of NoVs in shellfish
Aimed to identify a simple, rapid and highly efficient recovery method forreal-time RT-PCR detection of NoVs. Four methods were compared forrecovering GI.3and GII.4NoVs from spiked digestive tissues of oysters andclams, respectively. The method based on the application of proteinase K-PEG8000was found most efficient, with9.3%and13.1%of GI.3and GII.4NoVsrecovered from oysters and9.6%and12.3%of GI.3and GII.4NoVsrecovered from clams, respectively.
2. Presence of Genogroup II Norovirus in Retail Shellfish from Yellow seasand Bohai seas in China
Our objective was to evaluate the presence and contamination levels of NoVin shellfish sold at seafood markets in China. We tested840shellfish samples(Crassostrea gigas, Mytilus edulis, Azumapecten farreri, SinoNoVaculaconstricta, Scapharca subcrenata, Ruditapes philippinarum) that werecollected from seven cities around the Yellow and Bohai Seas in Chinabetween December2009and November2011. We used real-time RT-PCR todetect NoV in purified concentrates from the stomachand digestive diverticula of these shellfish. NoV was detected in19.35%(N=155),16.67%(N=114),5.70%(N=158),8.82%(N=136),13.74%(N=131), and16.44%(N=146) ofoyster, mussel, scallop, razor clam, ark shell, and clam samples, respectively.The average detection rate was13.33%(112/840). Nucleotide sequencing ofthe NoV RT-PCR products demonstrated that all strains belonged to NoVgenotype GII.12, except two that belonged to GI.3. More than102copies ofthe NoV genome were detected in69of112positive shellfish samples. Ourresults suggest that~13%of shellfish harbor NoV, and GII.12NoV is theprimary strain in shellfish purchased at markets in seven coastal cities inChina.
3. Risk assessment of NoVs in shellfish
Risk assessments of NoVs in shellfish were carried out based on the data ofinvestigation of NoVs presented in shellfish and shellfish diet survey. NoVsgastroenteritis infection probability of consumers along the coast areas ofChina by intake marine shellfish is4.08×10-7per person a day. The annualnumber of desease cases caused by consumption of polluted NoVs shellfish isabout229,17458and651among ages0-4,5-64and above65. Uncertaintyanalysis was also done in the study, which provides suggestion for NoVsassessment in shellfish or other food.
4. HBGAs detection in pacific oyster
The ELISA method based on was established for detection of A-type ofHBGAs in shellfish by optimized the concentration of heat treated oysterdigestive tisseues.36oyster samples were collected from Weihai, Qingdao andRizhao in2011. A-type of HBGAs and NoVs detection was analysed. Thedetection rate was100%and16.7%respectively. The results showed thatA-type of HBGAs changed along seasonal variation in fluctuating way.Interestingly, NoVs was detected at the time A-type of HBGAs showed peakvalue.
5. Prokaryotic expression and cloning of genes related to HBGAs in pacificoyster
Present on the oyster HBGAs synthesis of key gene has not been reported.With reference to the study of the synthetic pathway of human HBGAs, wedetermine the FUT genes for research purposes. By Clustal X and Mega andother analysis software, analysis of human, chimps, mice, dogs and otherspecies of the FUT2sequence, primers were designed based on conservedsequences of “FUT2”; The Pacific oyster (Crassostrea gigas) genomic DNAas the template for PCR amplification, cloning and sequencing the amplifiedproducts confirmed the existence in the oyster genome fragments from FUT2gene. In2012, with the completion of genome sequence of pacific oyster, the1081bp sequence we got was confirmed as FUT3. Prokaryotic expression ofFUT3was studied and We got the expected size of the protein.
6. Temperature effect on the expression regulation of FUT2in differenttissues of oyster
Real-time PCR detection method of FUT2genes in pacific oyster wasestablished with β-actin as the reference genes. FUT2genes expression inpacific oyster exposure to high temperature (28℃) and low temperature (4℃)was investigated by real-time quantitative PCR. The tissues analysed byquantitative real-time PCR were mantle, adductor, gill and digestive gland.Theexperiments were performed to investigate the expression level changes withinand between tissues at48h and72h.
The results indicated that level of most FUT2gene expression wassignificant higher than control no matter exposure to high temperature or lowtemperature conditions. When exposure to high temperature stress, level ofFUT2.1and FUT2.3genes expresstion have raised in4different tissues ofoyster organizations are raised, while under the stress of low temperature,FUT2.4expressed in mantle and gill tissue raised.
本文以贝类中NoVs为研究对象，优化建立了海产贝类中GI和GII型NoVs的富集方法，调查了黄渤海区7个城市零售海产贝类中NoVs的污染状况，在上述研究的基础上，结合CAC的食品安全风险分析理论，开展贝类中NoVs的风险评估研究；针对NoVs受体，建立了太平洋牡蛎中A型组织血型抗原的ELISA检测方法，并调查了威海、青岛、日照3个城市太平洋牡蛎样品中A型组织血型抗原随季节的变化规律；利用RACE技术得到了一个太平洋牡蛎组织血型抗原合成相关基因FUT3的cDNA全长，并进行了原核表达；利用牡蛎全基因组序列，初步探索了温度对牡蛎组织学型抗原合成关键基因FUT2的表达调控。具体研究结果如下：
1．贝类中NoVs富集方法的建立和优化
通过人工污染实验，向牡蛎和菲律宾蛤仔的消化道匀浆物中分别添加不同浓度的GI.3和GII.4型NoVs，利用4种贝类中NoVs富集方法分别回收病毒，用实验室前期建立的real-time RT-PCR方法进行检测。以4种方法从牡蛎和菲律宾蛤仔中富集GI.3和GII.4型NoVs的富集效率评价4种方法的优劣，同时分析影响NoVs富集的主要因素。结果显示方法B（Proteinase K-PEG8000）操作简单，耗时较短，且稳定性和灵敏性均优于其他方法，其从牡蛎和菲律宾蛤仔中富集GI.3型NoVs的富集效率分别为9.3%和9.6%，对GII.4型NoV的富集效率分别为13.1%和12.3%，比其他三种方法的富集效率高（p<0.05）。研究结果还发现贝类品种及贝类中NoVs的含量是影响贝类中NoVs富集效率的主要因素。
2．黄渤海贝类中NoVs污染状况调查
从2009年12月至2011年11月，采集了大连、莱州、烟台、威海、青岛、日照、连云港7个黄渤海区沿海城市6种零售贝类样品840个。调查结果显示NoVs在6种贝类中均有检出，平均检出率为13.33%(112/840)。不同贝类中NoVs的检出率为：牡蛎19.35%，紫贻贝16.67%，扇贝5.70%，缢蛏8.82%，毛蚶13.74%，菲律宾蛤仔16.44%。贝类中NoVs的季节分布结果显示，夏季贝类中NoVs检出率最高，为16.44%，冬季和秋季次之，春季最低。此外，研究发现不同季节NoVs检出率存在明显的品种特异性差异。春季和冬季牡蛎样品中NoVs的检出率最高，夏季毛蚶样品中NoVs的检出率最高，而冬季菲律宾蛤仔中NoVs的检出率最高。在冬季采集的33个扇贝样品中，NoVs无检出。
定量结果显示，112个阳性贝类样品中，96个样品中NoVs含量<103基因拷贝。运用spss17.0软件对不同贝类品种和不同季节贝类中NoV污染水平进行分析，非参检验（Kruskal-Wallis H tests）结果显示不同贝类品种和不同季节贝类中NoV污染水平均无明显差异(p>0.05)。112个阳性样品中，普通RT-PCR复检中仅96个样品琼脂糖凝胶电泳显示目的条带。将这96个样品测序结果输入Norovirus GenotypingTool Version1.0软件后，结果显示94个NoV属于GII.12变异株，2个属于GI.3。在冬季采集的菲律宾蛤仔和毛蚶中检测到GI.3型NoV。
3．贝类中NoVs的风险评估
本研究针对上述情况，利用前期调查研究获得的贝类中NoVs污染状况数据和相关膳食调查数据，对我国海产贝类中NoVs进行了定量风险评估，研究结果发现我国沿海城市居民食用贝类引发肠胃炎的平均概率为4.08E-7，预计每年0-4岁、5-64岁和65岁以上人口的发病数分别为119、9078和339人。本次评估只考虑贝类中NoVs污染率和污染水平以及贝类摄入量对风险的影响，存在一定的不确定性。国内外有关贝类中NoVs风险评估的研究较少，本次评估可以为贝类或其他食品中NoVs的风险评估提供一定的参考，相关调查资料可以为贝类食用安全提供指导意义。
4．牡蛎组织血型抗原的检测
在实验室前期研究的基础上改进前处理方法，建立了太平洋牡蛎中A型组织血型抗原的ELISA检测方法。运用建立的ELISA方法和第二章建立的贝类中NoVs的富集方法对2010年从威海、青岛、日照3个城市采集的36份太平洋牡蛎进行了A型组织血型抗原和NoVs的检测，结果表明三个城市太平洋牡蛎中A型组织血型抗原随季节变化呈现波动性变化，在全年不同月份出现峰值；36份牡蛎样品检出NoVs阳性6份，检出率为16.7%，且检出时间出现在A型组织血型抗原的高峰值处，推测贝类受NoVs污染可能与组织血型抗原表达相关。
5.牡蛎组织血型抗原相关基因的克隆及原核表达
参照人体组织血型抗原合成途径，选择FUT2基因作为牡蛎组织血型抗原合成关键基因。由于当时牡蛎全基因组序列尚未公布，通过Clustal X与Mega等分析软件，分析人、猩猩、小鼠、家犬等物种的FUT2基因氨基酸序列，推测牡蛎FUT2的保守区，根据保守序列设计引物。利用PCR方法扩增牡蛎组织血型抗原合成相关基因得到预期片段488bp的扩增产物，通过牡蛎“FUT2”扩增片段与人、小鼠、猩猩等物种的序列比对分析，可以初步证实牡蛎证实在牡蛎基因组中存在FUT2基因的保守片段。根据PCR方法得到的牡蛎“FUT2”的部分序列，设计引物，利用RACE扩增技术对牡蛎基因组进行扩增，克隆测序得到牡蛎“FUT2”基因3’和5’端序列，序列拼接后得到1086bp的cDNA全长基因。2012年9月，太平洋牡蛎全基因组序列公布，通过比对发现得到的牡蛎“FUT2”蛋白序列与FUT3基因同源性为98%，而与牡蛎FUT2基因的同源性为36%，因此证实所得序列为太平洋牡蛎FUT3基因。在此基础上，通过构建表达载体，成功诱导出FUT3的蛋白，大小为41.5kDa，为后续FUT3基因的功能验证打下基础。
6．温度对牡蛎组织血型抗原的表达调控
以β-actin作为内参基因，建立太平洋牡蛎中FUT2基因的荧光定量PCR检测方法。用高温（28℃）和低温（4℃）处理太平洋牡蛎，分别于48h和72h取样，研究牡蛎外套膜、闭壳肌、鳃和内脏4个组织中7组FUT2基因的表达情况。荧光定量PCR研究结果表明温度对FUT2部分基因的表达存在一定的影响。高温和低温条件，FUT2部分基因存在表达上调的现象。7组基因中，高温胁迫时FUT2.1和FUT2.3基因在牡蛎4个不同组织中表达上调；低温胁迫时，FUT2.4在外套膜和鳃组织中表达上调。
Noroviruses (NoVs) are commonly occurring pathogens that causegastroenteritis. NoVs can be isolated from water, vegetable, fruit andseafood samples, especially in shellfish samples frequently. It usually causesacute gastroenteritis associated with the consumption of raw or undercookedseafood. On rare occasion, infection can lead to severe dehydration and deathmay also occur subsequently. The annual production of shellfish was nearly upto10,000,000tons in China. For shellfish is delicious, the coastal residents arefond of it. Foodborne outbreaks caused by NoVs associated with theconsumption of shellfish were constantly reported in the coastal area of China.More attention should be paid to food safety issuses arised by NoVs.
To investigate the prevalence of NoVs in shellfish, the quantitative detectionmethods were optimized. The level of NoVs in shellfish of seven cities aroundYellow Sea and Bohai Sea in China was investigated by real-time RT-PCR andthen the quantitative assessment was carried out. The recent studies showedthat NoVs recognize histo-blood group antigens (HBGAs) as receptors inoyster.To elucidate the mechanism of NoVs accumulated in oysters, firstly, the ELISA and PCR method was optimized to detect A-type HBGAs and keygenes of HBGAs in oyster. Second, total length cDNA of one key genes FUT3was got and the protein was expressed.Temperature effect on expression levelof FUT2was also studied.The main contents were as follows:
1. The detection methods of NoVs in shellfish
Aimed to identify a simple, rapid and highly efficient recovery method forreal-time RT-PCR detection of NoVs. Four methods were compared forrecovering GI.3and GII.4NoVs from spiked digestive tissues of oysters andclams, respectively. The method based on the application of proteinase K-PEG8000was found most efficient, with9.3%and13.1%of GI.3and GII.4NoVsrecovered from oysters and9.6%and12.3%of GI.3and GII.4NoVsrecovered from clams, respectively.
2. Presence of Genogroup II Norovirus in Retail Shellfish from Yellow seasand Bohai seas in China
Our objective was to evaluate the presence and contamination levels of NoVin shellfish sold at seafood markets in China. We tested840shellfish samples(Crassostrea gigas, Mytilus edulis, Azumapecten farreri, SinoNoVaculaconstricta, Scapharca subcrenata, Ruditapes philippinarum) that werecollected from seven cities around the Yellow and Bohai Seas in Chinabetween December2009and November2011. We used real-time RT-PCR todetect NoV in purified concentrates from the stomachand digestive diverticula of these shellfish. NoV was detected in19.35%(N=155),16.67%(N=114),5.70%(N=158),8.82%(N=136),13.74%(N=131), and16.44%(N=146) ofoyster, mussel, scallop, razor clam, ark shell, and clam samples, respectively.The average detection rate was13.33%(112/840). Nucleotide sequencing ofthe NoV RT-PCR products demonstrated that all strains belonged to NoVgenotype GII.12, except two that belonged to GI.3. More than102copies ofthe NoV genome were detected in69of112positive shellfish samples. Ourresults suggest that~13%of shellfish harbor NoV, and GII.12NoV is theprimary strain in shellfish purchased at markets in seven coastal cities inChina.
3. Risk assessment of NoVs in shellfish
Risk assessments of NoVs in shellfish were carried out based on the data ofinvestigation of NoVs presented in shellfish and shellfish diet survey. NoVsgastroenteritis infection probability of consumers along the coast areas ofChina by intake marine shellfish is4.08×10-7per person a day. The annualnumber of desease cases caused by consumption of polluted NoVs shellfish isabout229,17458and651among ages0-4,5-64and above65. Uncertaintyanalysis was also done in the study, which provides suggestion for NoVsassessment in shellfish or other food.
4. HBGAs detection in pacific oyster
The ELISA method based on was established for detection of A-type ofHBGAs in shellfish by optimized the concentration of heat treated oysterdigestive tisseues.36oyster samples were collected from Weihai, Qingdao andRizhao in2011. A-type of HBGAs and NoVs detection was analysed. Thedetection rate was100%and16.7%respectively. The results showed thatA-type of HBGAs changed along seasonal variation in fluctuating way.Interestingly, NoVs was detected at the time A-type of HBGAs showed peakvalue.
5. Prokaryotic expression and cloning of genes related to HBGAs in pacificoyster
Present on the oyster HBGAs synthesis of key gene has not been reported.With reference to the study of the synthetic pathway of human HBGAs, wedetermine the FUT genes for research purposes. By Clustal X and Mega andother analysis software, analysis of human, chimps, mice, dogs and otherspecies of the FUT2sequence, primers were designed based on conservedsequences of “FUT2”; The Pacific oyster (Crassostrea gigas) genomic DNAas the template for PCR amplification, cloning and sequencing the amplifiedproducts confirmed the existence in the oyster genome fragments from FUT2gene. In2012, with the completion of genome sequence of pacific oyster, the1081bp sequence we got was confirmed as FUT3. Prokaryotic expression ofFUT3was studied and We got the expected size of the protein.
6. Temperature effect on the expression regulation of FUT2in differenttissues of oyster
Real-time PCR detection method of FUT2genes in pacific oyster wasestablished with β-actin as the reference genes. FUT2genes expression inpacific oyster exposure to high temperature (28℃) and low temperature (4℃)was investigated by real-time quantitative PCR. The tissues analysed byquantitative real-time PCR were mantle, adductor, gill and digestive gland.Theexperiments were performed to investigate the expression level changes withinand between tissues at48h and72h.
The results indicated that level of most FUT2gene expression wassignificant higher than control no matter exposure to high temperature or lowtemperature conditions. When exposure to high temperature stress, level ofFUT2.1and FUT2.3genes expresstion have raised in4different tissues ofoyster organizations are raised, while under the stress of low temperature,FUT2.4expressed in mantle and gill tissue raised.
引文
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