(bovine herpesvirus type 1, BHV1,. (infectious bovine rhinotracheitis, IBR) (infectious pustular vulvovaginitis, IPV) 3, 1.1, 1.2a 1.2b [5].

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1 : 3823 ~ csb.scichina.com SCIENCE IN CHINA PRESS gn,,,,,,, ;, cunzh2004@yahoo.com.cn , pha2 ZJ UL15 UL18, rbhv1-ha; DNA, DH10B, BHV1 pbhv1. pbhv1 MDBK,. Red E/T, gn pbhv1, rbhv1- gn., rbhv1- gn 9%~20%. BHV1, BHV-1. I gn UL49.5 (bovine herpesvirus type 1, BHV1), (infectious bovine rhinotracheitis, IBR) (infectious pustular vulvovaginitis, IPV).,,. BHV1 (bovine respiratory disease, BRD), BRD 10 [1] , [2,3]., BHV1 [3], [4] %, BHV1 α,, 3, 1.1, 1.2a 1.2b [5]. BHV bp, 73 (GenBank : NC_001847). 12, gb, gc, gd, ge, gg, gh, gi, gl, gm gk 10, gn gj [6]. BHV1 gn UL49.5, 9 kd, 96 [7,8]. (transporter associated with antigen processing, TAP), TAP,, MHC ;, gn, TAP [9,10]. gn BHV1,. :,,,. gn., 2009, 54: 3823~3829 Zhang C, Ye W C, Wang Y C, et al. Bovine herpesvirus type 1 genome cloned as infectious bacterial artificial chromosome and replication of gn gene deletion mutant in vitro (in Chinese). Chinese Sci Bull (Chinese Ver), 2009, 54: , doi: /

2 (bacteria artificial chromosome, BAC) 300 kb DNA,. BAC. MDV-1, EBV, EHV-1 BAC, BHV1 V155 Schonboken BAC [7,11~14]., BAC [15]. gn, BHV1 BAC; Red E/T gn, BHV1 gn. BHV1,. 1 ( ). BHV1 ZJ 2008, MDBK ; MDBK ATCC (CCL-22), 8% ( ) DMEM (Gibcol), 1% DMEM. pul15-18-ha2 ( 1(c)) Cornell,. BHV1 UL15 UL18, pha2 ( pbelobac11, Messerle M ) [16], GFP. ( ) BHV1 BAC. (1) DNA. 75 cm 2 MDBK, 10 MOI, 37 5%CO 2 70%~80%,, 700 g 5 min,, 4 10 mmol/l PBS (ph 7.4) 1, 500 µl TE (ph 8.0), 15 min; 5% NP µl RNase A 5 µl, 15 min; g 10 min,, 50 U ( ), min, EDTA. 0.5% SDS 0.2 mg/ml K, 52 2 h, - -, DNA, 50 µl TE. 5 µl Hind. (2). pul15-18-ha2 Qiagen Midiprep kit, Hind, DNA. 1 µg ZJ DNA 5 µg Hind pul15-18-ha2, ( Promega Calcium Phosphate Transfection Kit ) MDBK. 48 h, 60%,, 2.5% DMEM,. 3,, rbhv1-ha. (3) rbhv1-ha. [17]. 25 cm 2 MDBK, 0.1 MOI rbhv1-ha, 40%, PBS 1, 4000 g 5 min, 100 µl TE (ph 7.5, 10 mmol/l Tris, 1 mmol/l EDTA), 500 µl (ph 7.5, 10 mmol/l Tris, 0.6% SDS, 10 mmol/l EDTA) 5 µl RNase, 30 min, 400 µl 5 mol/l NaCl,, 4 24 h, g 30 min, - - 1,, DNA, 70%, 5 µl. (4) rbhv1-ha E. coli. rbhv1-ha DNA 50 µl E. coli DH10B 0.1 cm, 2500 V, 25 µf, 200 Ω (Bio-Rad ). 1 ml SOC, 37, 1 h, 34 µg/ml (CAM) LB, h,. (5) pbhv1. CAM 4.0 ml CAM LB, 37, DNA [18]. Hind, 0.8%, ; Sal,, BHV-1 ZJ BAC, pbhv1. ( ). Lipofectamine TM 2000 (Invitrogen). Qiagen Midi Kit pbhv1 DNA 1.0 µg ( pul µg) 250 µl opti-mem (Gibco); 10 µl Lipofectamine 250 µl opti-mem,, 15 min, 6 70% MDBK. 37 5% CO 2 6 h DMEM, 4 d, 80%, 3. BHV1-res. 3824

3 ( ) BHV1 gn. Red E/T [15,19] BHV1 ZJ gn. pbhv1 E. coli GS1783, Hind, pbhv1/ GS1783. pbhv1/gs1783 CAM LB, r/min A ~0.7, 42 30min Red, [17]. Red Kan gn. : gn-1stop-f gn-1stop-r ( 1) pepkans -Sce DNA ( -Sce -Kan), Qiagen, ; 100 ng pbhv1/gs1783, 32 1 h 34 µg/ml CAM 50 µg/ml Kan LB, 32 24~48 h., Sph (pbhv1- gn-kan). Red pbhv1- gn-kan Kan. : pbhv1- gn-kan, 2 ml CAM-LB, r/min 2~4 h, 2% L-, 1 h -Sce, min Red, 32 1 h ~10 5 CAM, 32 24~48 h, CAM Kan. Sph Cam Kan, (pbhv1- gn). pbhv1- gn, gn_seq-s gn_seq-as ( 1) PCR,. pbhv1- gn, ( ) rbhv1- gn. ( ). [13,14] BHV1, rbhv1-ha, rbhv1-res, rbhv1- gn. 24 MDBK, 1 TCID 50, 90 min, CBS (40 mmol/l, 10 mmol/l, 135 mmol/l, ph 3.0) 1, PBS 2, 1 ml. 2, 4, 6, 12, 4, h, 70. 1, 5000 r/min 5 min,, TCID 50, BHV1 BHV1 ZJ pul15-18-ha2, 1. pha2 pbelobac11, Pcmv GFP, GFP. BHV1 ZJ pul15-18-ha2 72 h,, 80%, MDBK, 3, rbhv1-ha ( 2). 2.2 pbhv1 rbhv1-ha MDBK 30 h, 40%, DNA, E. coli DH10B. CAM RLFP(Hind Sal ), 3. BHV1 ZJ pbhv1 BHV1-res, 1 BHV1 gn(ul49.5) a) b) gn-1stop-f 5 -CGAGGGGGCAATGGACTTTTGGAGCGCAGGCTGCTACGCGTAAAGGCTCATGGGCGCCAGCGGAGGATG ACGACGATAAGTAGGG-3 gn-1stop-r 5 -GCGACTCCTTTTTATTGGGCCCGCTGGCGCCCATGAGCCTTTACGCGTAGCAGCCTGCGCTCCCAACCAAT TAACCAATTCTGATTAG-3 gn _seq-s 5 -GTTTCATCTCGCTTTGCTGCTG-3 gn _seq-as 5 -CTGTTTTCTCGCACCCAAAGGTC-3 a) BHV1 GenBank NC_001847; b) pepkans ; BHV1 gn, (gn ), ( ) 3825

4 pha2 BHV-1 (a) BHV-1 Hind ; (b) pha2 DNA UL18 UL15 ; (c) BHV-1 pul15-18-ha2, 2 loxp pha2, 1.4 kb 2 rbhv-ha (a) ; (b). 1, 2 4, rbhv1-ha ; 3, BHV1. RLFP, BHV1 ZJ pbhv1, pha2, Hind Sal. 2.3 rbhv1- gn pbhv1- gn-kan ( Red ) pbhv1- gn ( Red ) Sph 4, gn ( ). Red, 6 pbhv1- gn,. 2 pbhv1- gn, gn_ seq-s gn _seq-as gn, gn, gn,. PCR, pbhv1- gn. pbhv1- gn MDBK 48 h,, rbhv1- gn. rbhv1- gn MDBK, gn BHV BHV1 ZJ, rbhv1-ha, rbhv1- res rbhv1- gn MDBK, 5., pbhv1 rbhv1-res. gn rbhv1- gn 9%~20%

5 3 BHV1 ZJ DNA pbhv1 Hind Sal. 1 5 BHV1, pha2 rbhv1-ha ( 2 6) ( ); rbhv1-ha pbhv1( 3 7),. 4 8 BHV1-res, pbhv1 pul15-18, BHV1 3 : (PRV) [20],, [21]. 4 pbhv1 gn Sph 1 pbhv1, RED Kan pbhv1- gn-kan( 2), pbhv1- gn-kan RED, Kan pbhv1- gn ( 3), pbhv1- gn RED. DNA ( M) GenScript Ladder 1997, (BAC), [22], HSV-1, Epstein-Barr (EBV) E. coli [16]. Timothy Trapp BHV1 V155 Schon- 5 BHV1 rbhv1-ha, rbhv1-res rbhv1- gn MDBK (a) ; (b) 3827

6 boken BAC [13,14]. BHV1 155, 1.2b, [13] ; Schonboken 1.2a, BHV1 ZJ, 1.2a,, Schonboken, BHV1 ZJ ; BHV1 BAC,, I. BHV1 BAC, Timothy Trapp TK ge BAC,, UL15 UL18 BAC, BHV1,. pbhv1 BAC, Red E/T, gn pbhv1- gn,,. BAC MDBK, gn BHV1. BHV1 gn TAP TAP [9,10], gn. gn BHV1, gn, gn ge. RecA- Red E/T, [15].,, ( ) [23,24]. BHV1 70, Red,, BHV1,. Cornell NiKlaus Osterrieder, Kui Yang Benedit Kaufer,,. 3828

7 24 Bovine herpesvirus type 1 genome cloned as infectious bacterial artificial chromosome and replication of gn gene deletion mutant in vitro ZHANG Cun 1, YE WeiCheng 1, WANG YiCheng 1, YUAN XiuFang 1, YIN LongBo 12, YIN WenLing 12 & LIU ManWen 1 1 Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou , China; 2 School of Veterinary Medicine, Northwest A & F University, Xi an , China The recombinant bovine herpesvirus type 1 (rbhv1-ha) was constructed by inserting pha2 plasmid into the viral genome between UL15 and UL18 cassettes. BHV1 infectious bacterial artificial chromosome had been confirmed after rbhv1-ha circular genome was extracted and transformed into E. coli strain DH10B. The resulting Bac clone, pbhv-1, was transfected into bovine kidney cells (MDBK) and the virus was rescued. The rescued virus, BHV1-res, have almost the same titres as the wildtype in replication in vitro. The transmembrane domain in gn open reading fram was deleted from pbhv-1 by Red E/T mutagenesis in E.coli. BHV-1 with partial deletion of gn, rbhv1- gn, was reconstituted from the mutated Bac. rbhv1- gn had lower titres by from 9% to 20% than rbhv-res in growth property in MDBK. This construction of infectious BHV1 clone should contribute greatly to the recombinant BHV1 vaccine with genes deletion and bovine universal viral vector. Bovine herpesvirus type 1, infectious clone, infectious bacterial artificial chromosome, membrane protein gn, UL49.5 doi: /