【作者】 林听听；

【导师】 战文斌； 邢婧；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2011， 博士

【摘要】 血细胞是贝类生物细胞免疫的主要执行者,是抵御外来病原侵袭和环境刺激的主要“屏障”。血细胞数量及胞内酶活的变动可以作为一个用于指示扇贝受病原感染或环境胁迫的免疫指标。本论文研究了栉孔扇贝（Chlamys farreri）经病毒感染和多糖刺激后血细胞数量及胞内酶活的变化规律;分类、分离了栉孔扇贝各种类型血细胞,以此为抗原研制出了抗各种类型血细胞的单克隆抗体;利用单抗制备了检测扇贝血细胞的酶联免疫（ELISA）试剂盒;应用试剂盒监测了扇贝在自然生长及胁迫环境下的血细胞变化;旨在为评估扇贝的健康状况提供手段和理论依据。本论文主要包括以下5部分:1栉孔扇贝经病毒感染后的血细胞变化用不同浓度(5⁰、5^-1、5^-2、5^-3、5^-4、5^-5、5^-6和5^-7)的栉孔扇贝急性病毒性坏死病毒（AVNV）,25℃（病发温度）人工感染扇贝,观察感染后各浓度组的存活率,确定了5^-3、5^-4和5^-5为较适宜的感染浓度。分别以5^-3和5^-5病毒浓度人工感染扇贝,感染后每天测定血细胞总数（THC）、血细胞内酸性磷酸酶（ACP）、碱性磷酸酶（ALP）、超氧化物歧化酶（SOD）、酚氧化酶（PO）、髓过氧化物酶（MPO）和过氧化物酶（POD）的变化,共测定15天。结果显示:感染后前8、9天,THC显著降低,而ACP、SOD、PO、MPO显著升高,POD无明显规律,ALP未被检测到;感染10天后,各免疫指标逐渐恢复到对照值。此外,5^-3浓度对各免疫指标的影响幅度要大于5^-5浓度。结果表明:血细胞在应激病毒感染过程中起着关键作用。2栉孔扇贝经β-葡聚糖刺激后的血细胞变化分别在15℃（最适温度）和25℃对栉孔扇贝注射0.5、1.0、2.0 mg/ml的β-葡聚糖,注射后每12小时测定THC和胞内ACP、ALP、SOD、PO、MPO和POD的变化,共测定7天。结果显示:15℃时,THC在0.5、1.0、2.0 mg/ml组均显著升高;而PO在0.5、1.0、2.0 mg/ml组,ACP,SOD和POD在1.0和2.0 mg/ml组,MPO和ALP分别在1.0和2.0 mg/ml组被显著激活。25℃时,THC只在1.0 mg/ml组显著升高;PO在0.5和1.0 mg/ml组,ACP在1.0和2.0 mg/ml组,SOD在1.0 mg/ml组被显著激活,而POD,MPO和ALP则未被激活或未被检测到。此外,THC、PO、ACP和SOD在25℃时的提升幅度和持续时间不及15℃。结果表明:血细胞在应激高温和β-葡聚糖刺激过程中起着关键作用;而高温能削弱扇贝识别和结合外源刺激物的能力,从而增加了被侵染的机率。3抗各种类型血细胞的单克隆抗体的研制用Percoll不连续密度梯度（10、20、30、40和50%）离心法分离了栉孔扇贝全血细胞,吉姆萨染色和流式细胞术鉴定了各层血细胞的成分,确定了30-40%界面层为透明细胞,而40-5⁰%界面层为颗粒细胞。分别收集透明细胞和颗粒细胞免疫小鼠,细胞融合,间接免疫荧光法（IIFA）筛选,克隆,所得单抗再经流式免疫荧光法（FCIFA）和Western blotting法（WBA）特性分析。结果显示:共筛选克隆得到7株既抗透明细胞和又抗颗粒细胞的单抗1F7,4D5,5C6,4G11,6A6,2H11,4E2,和1株只抗颗粒细胞的单抗6H7,没有得到只抗透明细胞的单抗。单抗1F7的阳性率为82±2.4%,其中阳性透明细胞（PH）占34±2.2%,阳性颗粒细胞（PG）占48±3.1%;它抗原决定簇丰富,能与多个血细胞蛋白结合。单抗4D5的阳性率为76±2.3%,其中PH占37±2.4%, PG占39±1.1%;它能与分子量为16.7,87和96 kDa的血细胞蛋白结合。单抗5C6的阳性率为70±2.6%,其中PH占32±1.9%,PG占38±2.4%;它能与分子量为16.7 kDa的血细胞蛋白结合。单抗6H7的阳性率为62±2.5%,其中PG占54±3.0%,PH仅占8±2.3%,它能与分子量为155 kDa的血细胞蛋白结合。4扇贝血细胞酶联免疫试剂盒的研制用IIFA,FCIFA和WBA从血细胞单抗库中筛选单抗作为栉孔扇贝血细胞酶联免疫试剂盒的第一抗体。梯度稀释抗原抗体,ELISA棋盘滴定法确定抗原抗体的最佳使用稀释度。高温（25℃）和病毒(5^-4AVNV)刺激栉孔扇贝,ELISA检测各刺激条件下的血细胞变化,以常温和高温刺激的比较结果制定扇贝正常状态与受环境胁迫的临界范围,以未感染和病毒感染的比较结果制定扇贝受环境胁迫与病原感染的临界范围,建立评估标准。最后组装制备试剂盒。结果显示:单抗1F7具阳性率高,抗透明细胞和颗粒细胞,及抗原决定簇丰富的特性被确定为试剂盒的第一抗体。抗原和抗体的最佳使用稀释度为抗原稀释2倍,抗体稀释4倍。待检样品OD_405nm值>0.57±0.025,属体质正常范围;待检样品OD_405nm值<0.47±0.019,属病原感染范围;两者之间为环境胁迫范围。试剂盒包括酶标板,封闭液,单抗1F7,碱性磷酸酶（AP）标记的羊抗鼠抗体,显色液,栉孔扇贝血细胞标准样品及检测结果评估标准等,其灵敏度可达200 ng/ml。IIFA分析了抗栉孔扇贝颗粒细胞的单抗6H7与虾夷扇贝（Patinopecten yessoensis）、海湾扇贝（Argopecten irradians）颗粒细胞的交叉性,确定了单抗6H7能与这两种扇贝的颗粒细胞特异性结合,但不与透明细胞结合。将单抗6H7选作为扇贝颗粒细胞酶联免疫试剂盒的第一抗体。分别梯度稀释栉孔扇贝、虾夷扇贝、海湾扇贝抗原,梯度稀释单抗6H7,ELISA棋盘滴定法确定抗原抗体的最佳使用稀释度。最后组装制备试剂盒。结果显示:不经稀释的抗原抗体具最佳的结合效果。试剂盒包括酶标板,封闭液,单抗6H7,AP标记的羊抗鼠抗体,显色液,三种扇贝血细胞标准样品等,其灵敏度可达5μg/ml。5扇贝血细胞酶联免疫试剂盒的应用于2009年3月至2010年1月,每月中旬从山东地区采集栉孔扇贝,测完壳长后,取血,用试剂盒研究栉孔扇贝血细胞变化与生长的关系。用5^-4AVNV人工感染栉孔扇贝,感染后用试剂盒每天测定颗粒细胞的变化,共测定9天。将海湾扇贝分成两组:一组每天实施投喂,另一组则不投喂,用试剂盒每周测定两组扇贝颗粒细胞的变化,共测定5周。结果显示:4,5,6月栉孔扇贝生长较快,对应的血细胞数量较多;8,9,10月生长缓慢,对应的血细胞数量则少。病毒感染后第1天,栉孔扇贝颗粒细胞数显著升高;随后急剧下降,于第3天达到最低值;之后开始回升,于第6、7天又显著升高;最后恢复到对照值。饥饿胁迫后,海湾扇贝颗粒细胞数前3周呈依次显著递减的趋势,3周后基本不再下降;投喂组的颗粒细胞数5周内基本维持同一水平。测定结果表明本论文研制的试剂盒具较好的应用效果。更多还原

【Abstract】 Bivalves frequently exposed to environmental stress and invasive or pathogenic microbes, and their survival depends to a large extent on the circulating cells collectively known as haemocytes. Haemocyte count and intracellular enzyme activity can be directly or indirectly used to evaluate the physiological state of a host or to estimate its immunity and potential defence capability against disease agents. In this dissertation, variations in haemocytes of scallop Chlamys farreri after viral and polysaccharidal stimulations were investigated. Then, C. farreri haemocytes were classified and separated. Subsequently, monoclonal antibodies （MAbs） against different types of C. farreri haemocytes were produced using the separated haemocytes as antigens. Afterwards, enzyme-linked immunosorbent assay （ELISA） kits for detection scallop haemocytes were prepared by employing these MAbs as primary antibodies. Finally, variations in scallop haemocytes in natural or stressed conditions were detected by ELISA kits. The main aim of this dissertation was to provide promising tool and theoretical basis for the assessment of health conditions of cultured scallops. This dissertation included the following 5 parts:1 Variation in haemocytes of C. farreri after a viral infectionDifferent concentrations (5⁰, 5^-1, 5^-2, 5^-3, 5^-4, 5^-5, 5^-6 and 5^-7) of acute viral necrobiotic virus （AVNV） were used to infect scallops C. farreri at 25°C. After infection, survival rates in each concentrations were counted, after that, among them, three concentrations of 5^-3, 5^-4 and 5^-5 were picked out as the desired infection concentrations. Subsequently, AVNV concentrations of 5^-3 and 5^-5 were used to infect scallops C. farreri at 25°C, and total haemocyte count （THC） and activities of acid phosphatase （ACP）, alkaline phosphatase （ALP）, superoxide dismutase （SOD）, myeloperoxidase （MPO）, phenoloxidase （PO） and peroxidase （POD） in haemocytes were measured every day till 15 days, respectively. The results showed that THC in viral groups was significantly lower than that in control group, while activities of ACP, SOD, MPO, PO were significantly higher than control group in the first 8 or 9 days, and went back to control group gradually after the 10^th day. Moreover, these five immune parameters in group of 5^-3 varied more than that in group of 5^-5. Differently, POD activity was reduced sometimes or induced other times, and showed no regular trends, while ALP activity wasn’t detected. The results clearly illustrated that THC and intracellular enzyme activities varied greatly after a viral infection, validating that haemocytes play a crucial role in the process of C. farreri against AVNV invasion.2 Variation in haemocytes of C. farreri after a polysaccharidal stimulationScallops C. farreri were treated with 0.5, 1.0 or 2.0 mg/ml ofβ-glucan suspensions at 15°C （the optimum temperature for C. farreri） or 25°C （the temperature at which the disease rate is elevated）, then THC, and the activities of PO, ACP, SOD, POD and ALP were measured in haemocytes every 12 h for seven days. The results showed that at 15°C, compared to the control, THC and PO in 0.5, 1.0 and 2.0 mg/ml, ACP, SOD and POD in 1.0 and 2.0 mg/ml, MPO and ALP respectively in 1.0 and 2.0 mg/mlβ-glucan were significantly increased. While at 25°C, THC and SOD in 1.0 mg/ml, PO in 0.5 and 1.0 mg/ml, ACP in 1.0 and 2.0 mg/mlβ-glucan were significantly induced, however, no significant induction of POD or MPO activity was observed, surprisingly, ALP activity was undetectable at 25°C throughout the entire experimental period. Moreover, the extent and duration of inducement as well as the time of peak emergence for these seven immune parameters appeared higher, longer and earlier at 15°C than 25°C in allβ-glucan treatments. These results clearly illustrated that THC and intracellular enzyme activities varied greatly, validating that haemocytes also play a crucial role in the process of C. farreri againstβ-glucan and high water temperature stresses, and high temperature may weaken or delay immune reactions to exogenous stress, thereby increasing the probability of being infected. 3 Production of MAbs against different types of C. farreri haemocytesScallop C. farreri haemocytes were separated by Percoll discontinuous density gradient （10, 20, 30, 40 and 50%） centrifugation, and the haemocytes at each resulting interface were collected and identified using flow cytometry and Giemsa staining, finding 30-40% interface of hyalinocytes, and 40-50% of granulocytes. Subsequently, separated hyalinocytes and granulocytes were respectively used as antigens to immunise mice. After cell fusion, screen using an indirect immunofluorescence assay （IIFA） and clone, the resulting MAbs were characterised using a flow cytometric immunofluorescence assay （FCIFA） and western blotting assay （WBA）, respectively. The results showed that more than fifty hybridomas secreting positive antibodies were obtained, however, most of the antibodies showed specificities for both hyalinocytes and granulocytes, such as MAb 1F7, 4D5, 5C6, 4G11, 6A6, 2H11 and 4E2, among them one MAb designated as 6H7 presented a specificity only for granulocytes, and no MAbs showed a specificity only for hyalinocytes. MAb 1F7 had a positive rate of 82±2.4%, among them, positive hyalinocytes （PH） of 34±2.2%, positive granulocytes of 48±3.1%, it had abundant epitopes, reacted with many haemocyte proteins. MAb 4D5 was 76±2.3%, PH of 37±2.4%, PG of 39±1.1%, it reacted with three haemocyte proteins of 16.7, 87 and 96 kDa. MAb 5C6 was 70±2.6%, PH of 32±1.9%, PG of 38±2.4%, it reacted with 16.7 kDa haemocyte protein. While MAb 6H7 was 62±2.5%, PG of 54±3.0%, PH of 8±2.3%, it reacted with 155 kDa haemocyte protein.4 Preparation of ELISA kits for detection scallop haemocytesSpecific MAb suitable for primary antibody of ELISA kit for detection C. farreri haemocytes was firstly screened out using IIFA, FCIFA and WBA. Then the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for antigen and screened MAb. Afterward, scallops C. farreri were stimulated by high water temperature （25°C） and 5^-4AVNV, respectively, and haemocyte variation in challenged scallops was detected by ELISA. Subsequently, an evaluation criteria was completed by finding a critical range between normal physical and environmental stress from the ELISA data comparison of normal temperature and high temperature group, a critical range between environmental stress and pathogenic infection from the ELISA data comparison of non-infected and infected groups. Finally, ELISA kit was assembled and prepared. The results showed that MAb 1F7 with a high positive rate, abundant epitopes, against both hyalinocytes and granulocytes, was suitable for primary antibody of ELISA kit for detection C. farreri haemocytes. The optimal using dilution of antigen and antibody was that antigen diluted two times, and antibody diluted four times. The critical range between normal physical and environmental stress was 0.57±0.025, between environmental stress and pathogenic infection was 0.47±0.019. This ELISA kit contained ELISA plate, blocking solution, MAb 1F7, alkaline phosphatase labeled goat anti-mouse antibody （AP-GMA）, colour developmental solution, standard samples and evaluation criteria, etc., with a sensitivity of 200 ng/ml.Granulocyte antigenic cross-reactivity of other two scallop species Patinopecten yessoensis and Argoecten irradians was carried out using MAb 6H7 against C. farreri granulocytes, illustrating MAb 6H7 also specifically reacted with the granulocytes of P. yessoensis and A. irradians, not with hyalinocytes, therefore, MAb 6H7 was chosen as primary antibody of ELISA kit for detection scallop granulocytes. After that, the optimal using dilution was found out using ELISA chessboard titration by respective gradient dilution for three antigens and MAb 6H7. Finally, ELISA kit was assembled and prepared. The results showed that not diluted antigen and antibody had a best reaction. This ELISA kit contained ELISA plate, blocking solution, MAb 6H7, AP-GMA, colour developmental solution and standard samples of three scallop species, etc., with a sensitivity of 5μg/ml.5 Application of ELISA kits for detection scallop haemocytesScallops C. farreri were collected monthly from Shandong area during the period from March 2009 to January 2010. After measurement of shell height, a relationship between haemocyte count and growth was investigated using ELISA kit. Scallops C. farreri were infected with 5^-4 AVNV, and granulocyte count was measured using ELISA kit every day for 9 days. Scallops A. irradians were divided into two groups: one group fed every day, the other one not fed, afterward, granulocyte count in two group was measured using ELISA kit every week for 5 weeks. The results showed that in April, May and June, C. farreri grew fast, correspondingly, in this period, C. farreri had a large number of haemocytes, while in August, September and October, C. farreri grew slowly, with a small number of haemocytes. After AVNV infection, C. farreri granulocytes count significantly increased on day 1, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8. After starvation stress, A. irradians granulocyte count significantly decreased in first 3 weeks, thereafter, decreased slightly, basically kept at a stable level, while in fed group, granulocyte count was maintained at the same level within 5 weeks. Conclusively, the results of these applications showed that ELISA kits developed in this dissertation had a promising prospect.更多还原