本發(fā)明屬于生物化學(xué)和分子生物學(xué)領(lǐng)域，具體涉及一種狂犬病毒抑制多肽及其制備方法和應(yīng)用。

背景技術(shù)：

狂犬病暴露后及時(shí)有效地處理是防治的關(guān)鍵，特別是狂犬病毒滅活疫苗和狂犬病毒抗體的聯(lián)合使用，是WHO推薦的人狂犬病暴露后預(yù)防的唯一選擇。即利用免疫手段使病毒在進(jìn)入細(xì)胞前就被消滅。一旦處理不及時(shí)，病毒就會(huì)入侵細(xì)胞，狂犬病的防治也就失去了最佳時(shí)機(jī)，從而造成嚴(yán)重后果。因?yàn)槟壳皩?duì)胞內(nèi)狂犬病毒的控制還沒(méi)有行之有效的方法。

利用siRNA技術(shù)抑制胞內(nèi)狂犬病毒的復(fù)制是一種很有前景的新方法，通過(guò)破壞或沉默病毒關(guān)鍵基因的表達(dá)，達(dá)到控制病毒復(fù)制的目的，該方法操作簡(jiǎn)單，效率高，毒性低，不足之處是缺乏安全、高效的胞內(nèi)運(yùn)輸系統(tǒng)。

磷蛋白是狂犬病毒的一種多功能蛋白，在致病過(guò)程中具有重要作用。作為一個(gè)必要中間體，通過(guò)與L蛋白和N蛋白結(jié)合，形成轉(zhuǎn)錄/復(fù)制復(fù)合體，參與病毒的轉(zhuǎn)錄和復(fù)制；除此之外，還可與宿主細(xì)胞的許多成分相互作用，如通過(guò)抑制IRF3和STAT1的活性，破壞宿主的先天免疫反應(yīng)；通過(guò)與PML蛋白結(jié)合，破環(huán)宿主的抗病毒機(jī)制等。因此磷蛋白是狂犬病防治的重要靶分子。Real E等采用酵母雙雜交系統(tǒng)從肽庫(kù)中篩選出一組多肽，可與磷蛋白結(jié)合從而干擾轉(zhuǎn)錄/復(fù)制復(fù)合物的形成，并通過(guò)細(xì)胞內(nèi)表達(dá)驗(yàn)證了其對(duì)病毒復(fù)制的抑制作用，為狂犬病毒抑制劑的開(kāi)發(fā)提供了重要的先導(dǎo)物。但其活性還有待進(jìn)一步提高，而且作為多肽藥物，胞內(nèi)給藥的方法仍然沒(méi)有解決。

針對(duì)以上問(wèn)題，設(shè)計(jì)出高活性、細(xì)胞透性好的多肽對(duì)于胞內(nèi)病毒的防治十分重要。定量構(gòu)效關(guān)系研究(quantitative structure-activity relationships，QSAR)是多肽分子優(yōu)化設(shè)計(jì)的常用方法，通過(guò)對(duì)一系列多肽的結(jié)構(gòu)或理化性質(zhì)的定量描述，并借助數(shù)學(xué)和統(tǒng)計(jì)學(xué)方法確定化學(xué)結(jié)構(gòu)與生物活性之間的關(guān)系，在此基礎(chǔ)上進(jìn)行新肽分子的活性預(yù)測(cè)和高活性肽的設(shè)計(jì)。

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明的目的是提供一種狂犬病毒抑制多肽，并通過(guò)對(duì)多肽序列的優(yōu)化達(dá)到提高病毒抑制效果的目的。

一種狂犬病毒抑制多肽，多肽序列如SEQ ID No.45所示：PPDVHTPPHALWRLHLILRVELVRMWWH。

進(jìn)一步地，為實(shí)現(xiàn)胞內(nèi)給藥，在胞內(nèi)抑制狂犬病毒的復(fù)制，在所述狂犬病毒抑制多肽的N端連接一段穿膜肽序列，所述穿膜肽序列可采用已知具備穿過(guò)細(xì)胞膜能力的短肽。

更進(jìn)一步地，所述穿膜肽序列為RRRRRRRRR，添加了穿膜肽的狂犬病毒抑制多肽序列如SEQ ID No.51所示：RRRRRRRRRPPDVHTPPHALWRLHLILRVELVRMWWH。

上述狂犬病毒抑制多肽的序列設(shè)計(jì)方法，采用QSAR方法，具體步驟如下：

(1)從已發(fā)表的文獻(xiàn)中搜集已被證實(shí)能抑制狂犬病毒復(fù)制的多肽數(shù)據(jù)用于建模，共計(jì)29條多肽，序列信息如SEQ ID No.1-29所示；

(2)通過(guò)PROFEAT服務(wù)器計(jì)算獲得29條多肽的描述符變量(http://bidd2.nus.edu.sg/cgi-bin/profeat2016/main.cgi)，分別選擇G3、G4、G5、G9四種描述符組合進(jìn)行計(jì)算；采用遺傳算法(Genetic algorithm，GA)篩選與多肽生物活性直接相關(guān)的理化參數(shù)，參數(shù)設(shè)置為：初始群體大小100、最大遺傳代數(shù)100、變異概率0.5％；

(3)采用偏最小二乘法(Partial least squares,PLS)建立QSAR模型，分別用Kennard stone、Euclidean距離及基于活性分組方法將樣本集分為訓(xùn)練集和測(cè)試集，用R²、Q²、和Q²_ext統(tǒng)計(jì)量評(píng)價(jià)模型的質(zhì)量，R²＞0.5、Q²＞0.5且Q²_ext＞0.5時(shí)認(rèn)為建模成功，作為后續(xù)分析基礎(chǔ)；

(4)從訓(xùn)練集或測(cè)試集中選擇活性值較高的多肽作為出發(fā)肽進(jìn)行優(yōu)化設(shè)計(jì)，進(jìn)一步提高其活性，分別對(duì)出發(fā)肽的每個(gè)氨基酸殘基進(jìn)行單點(diǎn)突變，并計(jì)算突變肽序列的描述符，選擇與訓(xùn)練集相同的描述符變量G3、G4、G5、G9進(jìn)行活性預(yù)測(cè)，活性高于出發(fā)肽的序列將是潛在的優(yōu)化多肽，可通過(guò)實(shí)驗(yàn)進(jìn)一步進(jìn)行篩選和活性驗(yàn)證。

進(jìn)一步地，上述狂犬病毒抑制多肽序列通過(guò)固相合成法或體外重組表達(dá)獲得。

本發(fā)明還提供了上述狂犬病毒抑制多肽在抑制狂犬病毒胞內(nèi)復(fù)制中的應(yīng)用。

進(jìn)一步地，所述應(yīng)用的劑量為12.5-50μg/ml培養(yǎng)液。

本發(fā)明的優(yōu)點(diǎn)：

1.本發(fā)明將QSAR法應(yīng)用于多肽的篩選及優(yōu)化設(shè)計(jì)，具有快速方便、針對(duì)性強(qiáng)、效率高的特點(diǎn)，可快速獲得活性提高的多肽序列。

2.本發(fā)明利用穿膜肽遞送藥物解決了多肽藥物入胞難的問(wèn)題。

3.本發(fā)明的狂犬病毒抑制多肽在胞內(nèi)發(fā)揮了抑制病毒復(fù)制的作用，抑制效果明顯，適用于狂犬病毒入胞后的處理，為入胞后病毒的控制提供了新的選擇。

4.本發(fā)明的優(yōu)化肽可作為先導(dǎo)物設(shè)計(jì)抑制病毒胞內(nèi)復(fù)制的小分子化合物。

附圖說(shuō)明

圖1帶穿膜肽的出發(fā)肽質(zhì)譜圖；

圖2帶穿膜肽的優(yōu)化肽質(zhì)譜圖；

圖3實(shí)施例2出發(fā)肽及優(yōu)化肽對(duì)狂犬病毒的抑制效果。

具體實(shí)施方式

為了使本發(fā)明的目的、技術(shù)方案及優(yōu)點(diǎn)更加清楚明白，以下結(jié)合附圖和實(shí)施例，對(duì)本發(fā)明進(jìn)行進(jìn)一步詳細(xì)說(shuō)明。應(yīng)當(dāng)理解，此處所描述的具體實(shí)施例僅用以解釋本發(fā)明，并不用于限定本發(fā)明。

下述實(shí)施例中所用到的BSR細(xì)胞、CVS毒株均由深圳市衛(wèi)光生物制品股份有限公司提供。

實(shí)施例1通過(guò)QSAR對(duì)狂犬病毒抑制多肽序列進(jìn)行優(yōu)化設(shè)計(jì)

(1)從已發(fā)表的文獻(xiàn)(Real E,Rain JC,Battaglia V,Jallet C,Perrin P,Tordo N,Chrisment P,D'Alayer J,Legrain P,Jacob Y.Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.J Virol.2004Jul；78(14):7410-7.)中搜集已被證實(shí)能抑制狂犬病毒復(fù)制的多肽數(shù)據(jù)用于建模，共計(jì)29條多肽，序列如表1所示：

表1 QSAR建模所用序列信息

(2)通過(guò)PROFEAT服務(wù)器計(jì)算獲得29條多肽的描述符變量(http://bidd2.nus.edu.sg/cgi-bin/profeat2016/main.cgi)，分別選擇G3、G4、G5、G9四種描述符組合進(jìn)行計(jì)算；采用遺傳算法(Genetic algorithm，GA)篩選與多肽生物活性直接相關(guān)的理化參數(shù)，參數(shù)設(shè)置為：初始群體大小100、最大遺傳代數(shù)100、變異概率0.5％；

(3)考慮到自變量可能存在多重相關(guān)性及樣本個(gè)數(shù)少于變量個(gè)數(shù)情況，采用偏最小二乘法(Partial least squares,PLS)建立QSAR模型，為了驗(yàn)證模型的外部預(yù)測(cè)能力，分別用Kennard stone、Euclidean距離及基于活性分組方法將樣本集分為訓(xùn)練集和測(cè)試集(見(jiàn)表1)，用R²、Q²、和Q²_ext統(tǒng)計(jì)量評(píng)價(jià)模型的質(zhì)量，R²≥0.998,Q²≥0.937，Q²_ext≥0.852，建模成功，本實(shí)施例中選擇活性分組方法建立的模型作為后續(xù)分析基礎(chǔ)；

(4)選擇活性值較高的SEQ ID No.18(活性為1.40)作為出發(fā)肽進(jìn)行優(yōu)化設(shè)計(jì)，進(jìn)一步提高其活性。分別對(duì)其序列中的28個(gè)氨基酸殘基進(jìn)行單點(diǎn)突變，并計(jì)算突變肽序列的描述符，選擇與訓(xùn)練集相同的描述符變量G3、G4、G5、G9進(jìn)行活性預(yù)測(cè)，經(jīng)預(yù)測(cè)表2中的序列活性明顯高于出發(fā)肽。

表2經(jīng)預(yù)測(cè)活性高于出發(fā)肽的突變肽序列

其中SEQ ID No.45(P16b6)的預(yù)測(cè)活性最高，因此作為優(yōu)化肽通過(guò)實(shí)驗(yàn)進(jìn)一步進(jìn)行篩選和活性驗(yàn)證。

實(shí)施例2出發(fā)肽及優(yōu)化肽的活性驗(yàn)證

將實(shí)施例1的出發(fā)肽及優(yōu)化肽通過(guò)固相合成法進(jìn)行合成，為了解決多肽藥物細(xì)胞透性差的問(wèn)題，分別在出發(fā)肽和優(yōu)化肽的N端連接一段穿膜肽序列RRRRRRRRR。所合成的帶穿膜肽的出發(fā)肽及帶穿膜肽的優(yōu)化胎的質(zhì)譜圖分別見(jiàn)圖1、2，序列分別為：

SEQ ID No.50：RRRRRRRRR PPDVHTPPHALWRLHLSLRVCLVRMWIH

SEQ ID No.51：RRRRRRRRRPPDVHTPPHALWRLHLILRVELVRMWWH。

平行準(zhǔn)備8組BSR細(xì)胞，分別以0.01MOI的CVS接種BSR細(xì)胞，1h后分別添加不同濃度的出發(fā)肽及優(yōu)化肽藥物(終濃度分別為0μg/ml、12.5μg/ml、25μg/ml、50μg/ml)，72h收獲上清液，測(cè)定病毒滴度。

如圖3所述，無(wú)論是出發(fā)肽還是優(yōu)化肽對(duì)病毒的復(fù)制均有抑制作用，并且表現(xiàn)出劑量效應(yīng)：濃度越高抑制效果越明顯。而優(yōu)化之后多肽的活性得到了明顯提高，也驗(yàn)證了穿膜肽作為多肽藥物載體的有效性。

應(yīng)當(dāng)理解的是，對(duì)本領(lǐng)域普通技術(shù)人員來(lái)說(shuō)，可以根據(jù)上述說(shuō)明加以改進(jìn)或變換，而所有這些改進(jìn)和變換都應(yīng)屬于本發(fā)明所附權(quán)利要求的保護(hù)范圍。

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<212> PRT

<213> Artificial Sequence

<220>

<223> 23

<400> 23

Pro Pro Asp Thr Ser Leu Leu Pro Pro Val Gly Leu His Leu Val Val

1 5 10 15

Arg Leu Phe Leu Leu Arg Leu Ser Val His

20 25

<210> 24

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 24

<400> 24

Pro Pro Gly Ala Pro Pro Ala Pro Phe Arg Thr His Thr Pro Pro Pro

1 5 10 15

Arg Met Val Ile Val Leu Ile Arg Val Trp Cys His

20 25

<210> 25

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 25

<400> 25

Pro Pro Gly Ala Pro Pro Gln Pro Asp Ser Val Cys Glu Leu His Leu

1 5 10 15

Leu Cys Val Leu Arg Leu Leu Val Ile Arg Ile His

20 25

<210> 26

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 26

<400> 26

Pro Pro Ser His Ser Phe Arg Pro Glu Ser Leu Glu Arg Leu His Leu

1 5 10 15

Leu Arg Arg Val Leu Leu Leu Met Arg Ile Val His

20 25

<210> 27

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 27

<400> 27

Pro Pro Cys Tyr Glu Arg Met Pro Arg Arg Leu Ile Arg Pro Pro Pro

1 5 10 15

Leu Leu Ser Val Leu Leu Ile Leu Arg Leu Cys His

20 25

<210> 28

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 28

<400> 28

Pro Pro Leu Phe Glu Asp Thr Pro Met Val Asn Ser Ile Pro Pro Leu

1 5 10 15

Arg Val Arg Leu Phe Leu Leu Arg Leu Val Phe His

20 25

<210> 29

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 29

<400> 29

Pro Pro Arg Gly Thr Glu Thr Pro Gln Arg Cys Arg Arg Leu His Leu

1 5 10 15

Val Glu Met Leu Cys Leu Val Arg Val Val Phe His

20 25

<210> 30

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 30

<400> 30

Pro Pro Asp Val His Phe Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Phe His

20 25

<210> 31

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 31

<400> 31

Pro Pro Asp Val His Trp Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Phe His

20 25

<210> 32

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 32

<400> 32

Pro Pro Asp Val His Thr Pro Phe His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Phe His

20 25

<210> 33

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 33

<400> 33

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu Gln Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Phe His

20 25

<210> 34

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 34

<400> 34

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Asp Leu Val Arg Met Trp Phe His

20 25

<210> 35

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 35

<400> 35

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Glu Leu Val Arg Met Trp Phe His

20 25

<210> 36

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 36

<400> 36

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Ile Leu Val Arg Met Trp Phe His

20 25

<210> 37

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 37

<400> 37

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Lys Leu Val Arg Met Trp Phe His

20 25

<210> 38

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 38

<400> 38

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Gln Leu Val Arg Met Trp Phe His

20 25

<210> 39

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 39

<400> 39

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met His Phe His

20 25

<210> 40

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 40

<400> 40

Pro Pro Asp Val His Phe Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Trp His

20 25

<210> 41

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 41

<400> 41

Pro Pro Asp Val His Trp Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Trp His

20 25

<210> 42

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 42

<400> 42

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu Gln Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met Trp Trp His

20 25

<210> 43

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 43

<400> 43

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Asp Leu Val Arg Met Trp Trp His

20 25

<210> 44

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 44

<400> 44

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Asp Leu Val Arg Met Trp Trp His

20 25

<210> 45

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 45

<400> 45

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Glu Leu Val Arg Met Trp Trp His

20 25

<210> 46

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 46

<400> 46

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Ile Leu Val Arg Met Trp Trp His

20 25

<210> 47

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 47

<400> 47

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Lys Leu Val Arg Met Trp Trp His

20 25

<210> 48

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 48

<400> 48

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Gln Leu Val Arg Met Trp Trp His

20 25

<210> 49

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> 49

<400> 49

Pro Pro Asp Val His Thr Pro Pro His Ala Leu Trp Arg Leu His Leu

1 5 10 15

Ile Leu Arg Val Cys Leu Val Arg Met His Trp His

20 25

<210> 50

<211> 37

<212> PRT

<213> Artificial Sequence

<220>

<223> 50

<400> 50

Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Asp Val His Thr Pro

1 5 10 15

Pro His Ala Leu Trp Arg Leu His Leu Ser Leu Arg Val Cys Leu Val

20 25 30

Arg Met Trp Ile His

35

<210> 51

<211> 37

<212> PRT

<213> Artificial Sequence

<220>

<223> 51

<400> 51

Arg Arg Arg Arg Arg Arg Arg Arg Arg Pro Pro Asp Val His Thr Pro

1 5 10 15

Pro His Ala Leu Trp Arg Leu His Leu Ile Leu Arg Val Glu Leu Val

20 25 30

Arg Met Trp Trp His

35