Gas-bearing fluid influx sub-system for gas hydrate geological system in Shenhu Area, Northern South China Sea

Based on the comprehensive interpretation and study of the Neogene fracture system and diapiric structure, it can be concluded that the diapiric structures, high-angle fractures and vertical fissure system are the main gas-bearing fluid influx sub-system for gas hydrate geological system in Shenhu Area, northern South China Sea. The Neogene fractures widely developed in the study area may be classed into two groups: NW (NNW)-trending and NE (NNE)-trending. The first group was active in the Late Miocene, while the second one was active since the Pliocene. The NE (NNE)-trending fractures were characterized by lower activity strength and larger scale, and cut through the sediment layers deposited since the Pliocene. Within the top sediment layers, the high-angle fracture and vertical fissure system was developed. The diapiric structures display various types such as a turtle-back-like arch, weak piercing, gas chimney, and fracture (or crack, fissure). On the seismic profile, some diapiric structures show the vertical chimney pathway whose top is narrow and the bottom is wide, where some ones extend horizontally into pocket or flower-shaped structures and formed the seismic reflection chaotic zones. Within the overlying sediment layers of the diapiric structure, the tree branch, flower-shaped high-angle fractures and vertical fissures were developed and became the pathway and migration system of the gas-bearing fluid influx. In the study area, the diapiric structures indicate a high temperature/over pressure system ever developed. Closely associated and abundant bright-spots show the methane-bearing fluid influx migrated vertically or horizontally through the diapiric structures, high-angle fractures and vertical fissures. In the place where the temperature and pressure conditions were favor for the formation of gas hydrate, the hydrate reservoir deposition sub-system was developed.

Efficient Simulation of Freak Waves in Random Oceanic Sea States

Numerical simulations of freak wave generation are studied in random oceanic sea states described by JONSWAP spectrum. The evolution of initial random wave trains is numerically carried out within the framework of the modified four-order nonlinear Schroedinger equation (mNLSE), and some involved influence factors are also discussed. Results show that if the sideband instability is satisfied, a random wave train may evolve into a freak wave train, and simultaneously the setting of the Phillips parameter and enhancement coefficient of JONSWAP spectrum and initial random phases is very important for the formation of freak waves. The way to increase the generation efficiency of freak waves though changing the involved parameters is also presented.

Methane hydrate growth velocity in seepage system of Qiongdongnan Basin, South China Sea

The occurrences of diapirs, gas-filled zones and gas plumes in seawater in Qiongdongnan Basin of South China Sea indicate that there may exist seepage system gas-hydrate reservoirs. Assuming there has a methane venting zone of 1500 m in diameter, and the methane flux is 1000 kmol/a, and the temperature of methane hydrate-bearing sediments ranges from 3 degrees C to 20 degrees C, then according to the hydrate film growth theory, by numerical simulation, this paper computes the temperatures and velocities in 0 mbsf, 100 mbsf, 200 mbsf, 425 mbsf over discrete length, and gives the change charts. The results show that the cementation velocity in sediments matrix of methane hydrate is about 0.2 nm/s, and the seepage system will evolve into diffusion system over probably 35000 years. Meanwhile, the methane hydrate growth velocity in leakage system is 20 similar to 40 times faster than in diffusion system.

SEA的基因克隆及高效表达工程菌的构建

Staphylococcal enterotoxin A(SEA）是由金黄色葡萄球菌分泌至胞外的一种单亚基蛋白质。它能与抗原递呈细胞上的MHC II受体结合，激发大量T细胞活化，释放多种细胞因子。正是由于SEA是一种强的T细胞激活剂，近年来，它在肿瘤的免疫治疗中显示出巨大的应用潜力。本文总结了国内外近十几年来在SEA治疗肿瘤方面的研究成果，指出，SEA靶向药物及低毒性SEA分子改造是SEA类药物研究的方向。利用PCR方法从金葡菌基因组DNA中扩增出seap基因（即SEA前体基因，含信号肤编码区），采用重叠PCR技术突变了694佩714碱基之间的所有稀有密码子，得到seap，。基因表达结果证实，改造seap基因中稀有密码子对提高基因表达水平非常有效。对seap基因中关键稀有密码子的突变在国内外尚属首次。以seapm为模板采用PCR方法扩增出seam (SEA成熟蛋白基因）。自行设计，自行构建成功一个新型原核表达载体7ZTS。它具有克隆，测序和表达在同一载体上进行，并可依据蓝白反应挑选重组子的特征。7ZTS表达载体将多种功能集于一体的独特优点是目前已知表达载体所不具备的，其大大简化了分子生物学操作步骤，提高了工作效率。利用7ZTS表达载体，在大肠杆菌JM109（DE3)中表达了mseap和seam基因。前者以包含体形式表达出SEA前体蛋白，后者以包含体和可溶性两种形式存在。通过均匀设计方法，摸索出'0sea基因表达的最佳条件为：诱导前生物量ODEo。为0. 8，装液量为28%，诱导温度为37'C，诱导时间为8小时。在此条件下，SEA表达量占细胞总蛋白的37%，可溶性SEA约占70%o SEA如此高效的表达为国内一外首次报道。质粒稳定性实验表明，7ZTS一msea重组质粒具有极好的稳定性，连续培养72小时或IPTG诱导48小时后，质粒基本不丢失。以上实验结果为SEA的大规模制备和SEA导向药物的研究开发奠定了基础。

抗黑色素瘤ScFv-超抗原SEA重组免疫毒素的构建与表达

提取鼠抗人黑色素瘤杂交瘤细胞株HB8759的总RNA，反转录成cDNA，用抗体可变区混合引物扩增出全套重、轻链可变区基因（VH、VLDNA），通过（Gly4Ser）3连接肤基因把VH和VL基因装配成单链抗体（ScFv）基因，将其克隆到噬菌粒载体pCANTABSE中，构建单链抗体噬菌体抗体库。用LIBr黑色素瘤细胞对抗体库进行了3轮亲合筛选。随机挑选克隆进行hage-ELISA鉴定，结果获得了2株具有较高ELISA活性的噬菌体单链抗体。序列测定证实得到的ScFv符合抗体可变区的结构特点，与已发表的鼠抗体可变区基因有较高的同源性。采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肤的金黄色葡萄球菌肠毒素A（SEA）基因进行融合，并将融合基因克隆于pET28-a表达载体的H招标签下游。SDS-PAGE分析表明，两种构建方法均表达了相对分子量约SOkD的蛋白条带，与预期目的蛋白分子量相符，主要以包涵体的形式存在，表达量占菌体蛋白的27％。将重组质粒在大肠杆菌中诱导表达，用盐酸肌溶解包涵体，Ni-NTA鳌合层析柱一步法纯化包涵体，再通过透析使目的蛋白复性。凝胶灰度扫描显示蛋白纯度达90％，凝胶电泳呈单一条带，蛋白量达0.47mg／mL。用健康人外周血单个核细胞作为效应细胞，LDH法检测ScFv-SEA融合蛋白对LiBr黑色素瘤细胞、MCF7乳腺癌的体外抑制率。结果表明该融合蛋白可以通过活化效应细胞，对表达相关抗原的肿瘤细胞产生有效的抗增殖作用，而对不表达该抗原的肿瘤细胞作用不显著。说明该融合蛋白赋予了SEA抗黑色素瘤特异性。以上实验结果为我们研究SEA对黑色素瘤的靶向杀伤作用奠定了可靠的实验基础。