兔抗人乳酸脱氢酶C4(LDHC4)多克隆抗体制备与应用

目的 制备兔抗人乳酸脱氢酶C4(LDHC4)的多克隆抗体。方法 采用PCR点突变LDHC基因,并将该基因构建到pET-28a载体上,形成pET-28a-LDHC重组表达载体、将重组载体转化至大肠杆菌表达宿主BL21(DE3),诱导表达获得重组人LDHC4蛋白,将重组蛋白作为抗原免疫新西兰兔,3次加强免疫后得到抗血清,采用ELISA检测抗血清效价,采用Western blot法检测抗血清的特异性,采用免疫组织化学法检测人三阴性乳腺癌组织中LDHC4的表达。结果 获得了特异性的兔抗人LDHC4多克隆抗体,抗体效价达1∶51 200,该抗体可用于Western blot法和免疫组织化学法。结论 成功制备了特异性兔抗人LDHC4多克隆抗体。

Immuno-contraceptive potential of sperm specific LDHC₄and SPAM-1 (PH-20) sub units in dog

PH-20 (Spam-1) antigen appears to be a bi-functional sperm plasma membrane protein with hyaluronidase activity and its role is related to cumulus penetration as well as zona binding. (LDH-C4) is a key enzyme, distributed specifically in testis and is highly immunogenic, function of which relates to energy metabolism and capacitation of the sperm. Therefore, in this study, we observed the effect of purified native PH-20 and LDH-C4 antigens on sperm function and morphology in dog. Native PH-20 (46, 32 kDa) and LDH-C4 (36, 30, 28) sub units, purified from dog sperm SDS- extracts were used to test as immunocontraceptive candidates in stray dog. Dogs 1, 2, 3, 4, 5/6 and 7/8 were immunized with 46, 32, 36, 30/28, 46/32, 36/30/28 kDa sub units respectively by i/m route and semen was analysed at weekly intervals. High titre of 3200 - 6400 was achieved in all dogs after three boosters. Immune response in treated dogs was confirmed by increase in Ig level in blood serum and reaction of partially purified serum Ig of immunized dogs with respective antigens in double immune diffusion and immunoblots. Immunization of dogs either with individual PH-20/ LDHC sub units or pooled PH-20/LDHC4 sub units showed a significant effect on sperm count, percent motility, viability, HOST and sperm morphology. All the sperm parameters declined to a significant level (p < 0.05) between 45 - 115 DPI and remained low than that of the recommended values for fertile dog semen till 175 DPI. The incubation of spermatozoa in HOS solution in the presence of anti-PH-20 and anti-LDHC Ig significantly reduced the percent HOS positive spermatozoa. A significant decline (p ≤ 0.05) in acrylamide penetration assay was also observed in the presence of anti 46 and 30/28 kDa Ig. Since percent motility, sperm count, HOST and percent normal spermatozoa in immunized dogs were significantly less than that of recommended fertile dog semen, it can be concluded that PH-20 (46/32 kDa) and LDHC4 (36/30/28 kDa) sub units had an immunocontraceptive potential in dog.

Identification of human LDHC4 as a potential target for anticancer drug discovery

One of the distinct hallmarks of cancer cells is aerobic glycolysis(Warburg effect). Lactate dehydrogenase A(LDHA) is thought to play a key role in aerobic glycolysis and has been extensively studied, while lactate dehydrogenase C(LDHC), an isoform of LDHA, has received much less attention.Here we showed that human LDHC was significantly expressed in lung cancer tissues, overexpression of Ldhc in mice could promote tumor growth, and knock-down of LDHC could inhibit the proliferation of lung cancer A549 cells. We solved the first crystal structure of human LDHC4 and found that the activesite loop of LDHC4 adopted a distinct conformation compared to LDHA4 and lactate dehydrogenase B4(LDHB4). Moreover, we found that(ethylamino)(oxo)acetic acid shows about 10 times selective inhibition against LDHC4 over LDHA4 and LDHB4. Our studies suggest that LDHC4 is a potential target for anticancer drug discovery and(ethylamino)(oxo)acetic acid provides a good start to develop lead compounds for selective drugs targeting LDHC4.

雷公藤服用者精液中乳酸脱氢酶及其同功酶C_4活性的研究

本文对19例中药雷公藤服用者精子、精浆中乳酸脱氢酶(LDH)及同功酶C_4(LDHC_4)的活性进行了测定分析。发现服药后以单位数量精子表示的LDHC_4活性在精子、精浆中均有明显升高(双侧非参数秩和检验,分别为P<0.01和P<0.02);通过酶活性与精子计数的相关分析,显示服药后精浆LDHC_4活性(mU/ml)与精子计数的正相关性(r=0.2279)较服药前(r=0.5505)弱,可能与精液中精子以外的其他LDHC_4来源贡献增大有关,诸如生精细胞的分解增多等。本文认为服药后精子LDHC_4活性上升,精液中非成熟精子比例增高是其原因之一。