摘要
近年来，昆虫抗菌肽的研究逐渐成为昆虫免疫学及分子生物学研究热点
一，人们希望在不久的将来，抗菌肽能替代传统的抗生素而成为新一代抗菌剂
和抗肿瘤新药。然而对于昆虫体液免疫的另一个重要组成部分——昆虫抗病毒
的研究却一直没有新的突破。为了深入研究昆虫抗病毒免疫的机理，本文探讨
了虫体及昆虫离体细胞在感染病毒后能否产生病毒抑制因子及抗病毒活性，同
时对昆虫抗菌活性物质的抗病毒活性进行了检测。主要结果如下：
用BmNPV感染家蚕幼虫，取其免疫血淋巴用（NH_4）_2SO_4分级沉淀，
制得粗VIF样品Ⅰ和样品Ⅱ。同时以健康家蚕血淋巴提取物为对照组。将含有
AcMNPV（5×10~6 TCID_(50)/ml）的细胞上清与等体积粗VIF样品混合温育1h后，
感染对数生长期5B1细胞。4d后观察发现，诱导组内少数细胞出现核膨大，
核内出现多角体的细胞数目较少；而对照组中细胞病变明显，核内出现多角体
的细胞数目多，核极端膨大，核内充满多角体，有的细胞破裂并释放出多角体。
对粗VIF样品Ⅰ和样品Ⅱ进行抗病毒活性测定，灭活指数分别为1.99、2.33。
将粗VIF样品Ⅰ和样品Ⅱ上DEAE-Sepharose离子交换柱初步纯化，活性峰分
别为峰C_1、峰b_2。峰C_1、峰b_2经PEG20000浓缩后进行抗病毒活性测定其灭
活指数分别为2.17、2.50。对粗VIF样品Ⅰ和样品Ⅱ进行SDS-PAGE电泳分析
表明粗VIF样品Ⅰ比对照样品Ⅰ多出四条带，它们的分子量分别为：83.7KD、
65.2KD、53.3KD、32.8KD；粗VIF样品Ⅱ比对照样品Ⅱ多出一个组分并且
有三个组份表达量增多，多出的组分分子量为36.0KD。分析灭活指数最高的
峰b_2，存在五条带，其中有一条带对应于样品Ⅱ上多出的那个组分。因此初步
卜自氨习 硕士兰垃论丈
证明感染BmWV的家蚕的免疫血淋巴中产生了VIF。
用紫外灭活的 A。MN’PV诱导离体 SBI细胞，粗提物未检测到明显的抗病
毒活性。分别用紫外灭活 Ac删V 10 min、15 Inn、20 Inn、25 min及 30 Inn
后，感染 SBI细胞，将细胞上清液浓缩，粗提物分别进行抗病毒活性检测，灭
活指数分别为：－0二1、－0．18、＋0．17、＋0．31、＋0二7。
用热灭活的大肠杆菌DHS。诱导SBI细胞，处理12h后观察，大量细菌均
匀沉积在细胞周围，细胞有明显的拉网现象，细胞生长状态差；处理24h后观
察，部分细菌被细胞吞噬进去；处理48小时后，细胞表面的细菌有明显减少，
细胞生长状态逐渐正常。取处理 16 h后的细胞诱导上清浓缩后，测其抗菌活性，
与正常细胞提取物对照相比，含有诱导上清提取物的滤纸片周围形成了抑菌
圈，证明诱导上清有抗菌活性物质存在。但用此诱导上清提取物做抗病毒活性
测定，灭活指数为0．5，因此认为此抗菌活性物质不具有明显抗病毒活性。
Recently, the research on antibacterial peptites from insects becomes a key
concern in the field of insect immunobiology and molecular biology It has been
expected that antibacterial peptites will be used as a new type of antibiont and a
new anticancer drug. However, few breakthroughs have been made in the research
of insect antiviral immunity which plays an important role in the insect humoral
immunity. For further study, in the paper the question as to whether the VIF could
be induced in the hemolymph of silkworm larvae and cultured insect cell lines
infected with baculovirus was discussed, their antiviral activities were identified
and the antiviral activity of insect antibacterial protein was tested as well. The
results as follows;
1. The bemolymph of silkworm larvae infected with BmNPV was fractionated
gradedly with amznonium sulfate. AcM7NPV was mixed with an equal volume of
crude VIF and incubated at 28 0C for 60 mm. The SB 1 cells were infected with two
mixtures, Crude VIE I and II, respectively. Four days later, we observed that few
cells were infected, compared with the control. And inactivated the virus strongly
since the inactivation indexes of them were 1.99 and 2.33, respectively.
Crude VIE I and II were further purified with DEAE-Sepharose Fast Flow
chromatography and emerged active peak Ci and peak b2 containing the antiviral
protein, respectively. They inactivated the virus strongly since the inactivation
indexes of peak c1 and peak b2 were 2.17 and 2.50, respectively. Then crude VIF I
and II were subjected to SDS-PAGE analysis. Crude VIF I was found to
produce four new bands, and the molecular weight of them were:83.7KD,
65.2KD,53.3KD,32.8KD,while crude VIF Ilwas found to produce a single new
band with the molecular weight of 36.OKD and three enhanced bands. Five bands
were observed in the peak b2, one of which is similar to the 36.OKD band produced
iii
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by the crude VIE II. Therefore, the results indicated that viral inhibitory factor was
induced the hemolymth of silkworm larvae infected with BmINPV.
2. The 5B1 cells were infected with ultra-violet inactivated AcMNPV in 10
mm, 1 5mm, 20 mm, 25 mm and 30 mm, respectively. Their antiviral activities were
tested, and their inactivation indexes were ?.21, -0.18, -I-U. 17, ?.31 and 0.13,
respectively. We supposed that the antiviral activity couldn抰 be induced in cultured
insect cells.
3. The 5B1 cells were treated with heat-killed E. Coli strain DH5 .The
antibacterial activity could be detected by measuring the zone of growth inhibition.
However, How most people thought the limit value of the antiviral activity was 0.7,
and we found the inactivation index of the antibacterial activity was 0.5, we
concluded that the antibacterial agent had no antiviral activity.
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